Regulation of cell-matrix adhesion by OLA1, the Obg-like ATPase 1.

Attachment of cells to the extracellular matrix induces clustering of membrane receptor integrins which in turn triggers the formation of focal adhesions (FAs). The adaptor/scaffold proteins in FAs provide linkage to actin cytoskeleton, whereas focal adhesion kinase (FAK) and other FA-associated kinases and phosphatases transduce integrin-mediated signaling cascades, promoting actin polymerization and progression of cell spreading. In this study, we explored the role of OLA1, a newly identified member of Obg-like ATPases, in regulating cell adhesion processes. We showed that in multiple human cell lines RNAi-mediated downregulation of OLA1 significantly accelerated cell adhesion and spreading, and conversely overexpression of OLA1 by gene transfection resulted in delayed cell adhesion and spreading. We further found that OLA1-deficient cells had elevated levels of FAK protein and decreased Ser3 phosphorylation of cofilin, an actin-binding protein and key regulator of actin filament dynamics, while OLA1-overexpressing cells exhibited the opposite molecular alterations in FAK and cofilin. These findings suggest that OLA1 plays an important negative role in cell adhesion and spreading, in part through the regulation of FAK expression and cofilin phosphorylation, and manipulation of OLA1 may lead to significant changes in cell adhesion and the associated phenotypes.

Unraveling the rapid CO2 mineralization experiment using the Paraná flood basalts of South America.

CO2 capture and storage in geological reservoirs have the potential to significantly mitigate the effects of anthropogenic gas emissions on global climate. Here, we report the results of the first laboratory experiments of CO2 injection in continental flood basalts of South America. The results show that the analyzed basalts have a mineral assemblage, texture and composition that efficiently allows a fast carbonate precipitation that starts 72 h after injection. Based on the availability of calcium, chemical monitoring indicates an estimated CO2 storage of ~ 75%. The carbonate precipitation led to the precipitation of aragonite (75.9%), dolomite (19.6%), and calcite (4.6%).

First report of the prevalence of Shiga toxinproducing Escherichia coli in ground beef in Quindío, Colombia. / Primer reporte de la prevalencia de Escherichia coli productora de toxina Shiga en carne molida en Quindío, Colombia

INTRODUCTION: Shiga toxin-producing Escherichia coli (STEC) is a foodborne pathogen associated with clinical cases of diarrhea in humans. Its main virulence factors are the Shiga toxins (Stx1 and Stx2). Cattle are the main reservoir of STEC, and many outbreaks in humans have been related to the consumption of undercooked ground beef contaminated with this pathogen. OBJECTIVE: To determine the prevalence of STEC in ground beef commercialized in all the butcher shops of a township in the department of Quindío and to characterize the virulence genes of the strains found. MATERIALS AND METHODS: Thirty ground beef samples were taken in three different times; stx genes and other STEC virulence factors (eae, ehxA, saa) were detected by multiplex PCR. RESULTS: The overall prevalence of STEC was 33.33 % (10/30 positive samples). We isolated eight non-O157 (LEE-negative) strains with four different genetic profiles: stx2 / stx2-ehxA-saa / stx1-stx2-ehxA-saa / stx1-saa. CONCLUSION: This is the first report on the prevalence of STEC in ground beef in a township in the department of Quindío.

Introducción: Escherichia coli productora de toxina Shiga (STEC) es un agente patógeno de origen alimentario asociado a casos clínicos de diarrea en humanos; sus principales factores de virulencia son las toxinas Shiga (Stx1 y Stx2). El principal reservorio de STEC es el ganado bovino y muchos brotes en humanos se han relacionado con el consumo de carne mal cocida contaminada con este agente patógeno. Objetivo: El objetivo de este trabajo fue determinar la prevalencia de STEC en carne molida comercializada en todas las carnicerías de un municipio del departamento del Quindío y caracterizar los genes de virulencia de las cepas encontradas. Materiales y métodos: Se tomaron 30 muestras de carne molida en tres momentos diferentes; se detectaron los genes stx y otros factores de virulencia de STEC (eae, ehxA, saa) mediante PCR Multiplex. Resultados: Los resultados mostraron una prevalencia global de STEC del 33,33 % (10/30 muestras positivas). En total se aislaron ocho cepas STEC no-O157 (LEE-negativas) con cuatro perfiles genéticos diferentes: stx2 / stx2-ehxA-saa / stx1-stx2-ehxA-saa / stx1-saa. Conclusión: Este es el primer reporte que muestra la prevalencia de STEC en carne molida en un municipio del departamento del Quindío.

Arable soil nitrogen dynamics reflect organic inputs via the extended composite phenotype.

Achieving food security requires resilient agricultural systems with improved nutrient-use efficiency, optimized water and nutrient storage in soils, and reduced gaseous emissions. Success relies on understanding coupled nitrogen and carbon metabolism in soils, their associated influences on soil structure and the processes controlling nitrogen transformations at scales relevant to microbial activity. Here we show that the influence of organic matter on arable soil nitrogen transformations can be decoded by integrating metagenomic data with soil structural parameters. Our approach provides a mechanistic explanation of why organic matter is effective in reducing nitrous oxide losses while supporting system resilience. The relationship between organic carbon, soil-connected porosity and flow rates at scales relevant to microbes suggests that important increases in nutrient-use efficiency could be achieved at lower organic carbon stocks than currently envisaged.

OLA1, an Obg-like ATPase, suppresses antioxidant response via nontranscriptional mechanisms.

Oxidative stress has been implicated in diverse disease states and aging. To date, induction of cellular responses to combat oxidative stress has been characterized largely at the transcriptional level, with emphasis on Nrf2-mediated activation of antioxidant response elements. In this study, we demonstrate that OLA1, a novel Obg-like ATPase, functions as a negative regulator of the cellular antioxidant response independent of transcriptional processes. Knockdown of OLA1 in human cells elicited an increased resistance to oxidizing agents including tert-butyl hydroperoxide (tBH) and diamide without affecting cell proliferation, baseline apoptosis, or sensitivity to other cytotoxic agents that target the mitochondria, cytoskeleton, or DNA. Conversely, overexpression of OLA1 increased cellular sensitivity to tBH and diamide. When challenged with oxidants, OLA1-knockdown cells had decreased production of intracellular reactive oxygen species and exhibited less depletion of reduced glutathione. Surprisingly, knockdown of OLA1 caused only minimal genomic response; no changes were found in the mRNA levels of genes encoding antioxidant enzymes, enzymes that produce antioxidants (including glutathione), or other genes known to respond to Nrf2. Moreover, when de novo protein synthesis was blocked by cycloheximide in OLA1-knockdown cells, they continued to demonstrate increased resistance to both tBH and diamide. These data demonstrate that OLA1 suppresses the antioxidant response through nontranscriptional mechanisms. The beneficial effects observed upon OLA1-knockdown suggest that this regulatory ATPase is a potential novel target for antioxidative therapy.

First report of the prevalence of Shiga toxin-producing Escherichia coli in ground beef in Quindío, Colombia / Primer reporte de la prevalencia de Escherichia coli productora de toxina Shiga en carne molida en Quindío, Colombia

Introduction. Shiga toxin-producing Escherichia coli (STEC) is a foodborne pathogen associated with clinical cases of diarrhea in humans. Its main virulence factors are the Shiga toxins (Stx1 and Stx2). Cattle are the main reservoir of STEC, and many outbreaks in humans have been related to the consumption of undercooked ground beef contaminated with this pathogen. Objective. To determine the prevalence of STEC in ground beef commercialized in all the butcher shops of a township in the department of Quindío and to characterize the virulence genes of the strains found. Materials and methods. Thirty ground beef samples were taken in three different times; stx genes and other STEC virulence factors (eae, ehxA, saa) were detected by multiplex PCR. Results. The overall prevalence of STEC was 33.33 % (10/30 positive samples). We isolated eight non-O157 (LEE-negative) strains with four different genetic profiles: stx 2 / stx 2-ehxA-saa / stx 1-stx 2-ehxA-saa / stx 1-saa. Conclusion. This is the first report on the prevalence of STEC in ground beef in a township in the department of Quindío.

Introducción. Escherichia coli productora de toxina Shiga (STEC) es un agente patógeno de origen alimentario asociado a casos clínicos de diarrea en humanos; sus principales factores de virulencia son las toxinas Shiga (Stx1 y Stx2). El principal reservorio de STEC es el ganado bovino y muchos brotes en humanos se han relacionado con el consumo de carne mal cocida contaminada con este agente patógeno. Objetivo. El objetivo de este trabajo fue determinar la prevalencia de STEC en carne molida comercializada en todas las carnicerías de un municipio del departamento del Quindío y caracterizar los genes de virulencia de las cepas encontradas. Materiales y métodos. Se tomaron 30 muestras de carne molida en tres momentos diferentes; se detectaron los genes stx y otros factores de virulencia de STEC (eae, ehxA, saa) mediante PCR Multiplex. Resultados. Los resultados mostraron una prevalencia global de STEC del 33,33 % (10/30 muestras positivas). En total se aislaron ocho cepas STEC no-O157 (LEE-negativas) con cuatro perfiles genéticos diferentes: stx 2 / stx 2-ehxA-saa / stx 1-stx 2-ehxA-saa / stx 1-saa. Conclusión. Este es el primer reporte que muestra la prevalencia de STEC en carne molida en un municipio del departamento del Quindío.

OLA1, a Translational Regulator of p21, Maintains Optimal Cell Proliferation Necessary for Developmental Progression.

OLA1, an Obg-family GTPase, has been implicated in eukaryotic initiation factor 2 (eIF2)-mediated translational control, but its physiological functions remain obscure. Here we report that mouse embryos lacking OLA1 have stunted growth, delayed development leading to immature organs-especially lungs-at birth, and frequent perinatal lethality. Proliferation of primary Ola1(-/-) mouse embryonic fibroblasts (MEFs) is impaired due to defective cell cycle progression, associated with reduced cyclins D1 and E1, attenuated Rb phosphorylation, and increased p21(Cip1/Waf1) Accumulation of p21 in Ola1(-/-) MEFs is due to enhanced mRNA translation and can be prevented by either reconstitution of OLA1 expression or treatment with an eIF2α dephosphorylation inhibitor, suggesting that OLA1 regulates p21 through a translational mechanism involving eIF2. With immunohistochemistry, overexpression of p21 protein was detected in Ola1-null embryos with reduced cell proliferation. Moreover, we have generated p21(-/-) Ola1(-/-) mice and found that knockout of p21 can partially rescue the growth retardation defect of Ola1(-/-) embryos but fails to rescue them from developmental delay and the lethality. These data demonstrate, for the first time, that OLA1 is required for normal progression of mammalian development. OLA1 plays an important role in promoting cell proliferation at least in part through suppression of p21 and organogenesis via factors yet to be discovered.

OLA1 contributes to epithelial-mesenchymal transition in lung cancer by modulating the GSK3ß/snail/E-cadherin signaling.

Obg-like ATPase 1 (OLA1) belongs to the Obg family of P-loop NTPases, and may serve as a "molecular switch" regulating multiple cellular processes. Aberrant expression of OLA1 has been observed in several human malignancies. However, the role of OLA1 in cancer progression remains poorly understood. In this study, we used the Kaplan-Meier plotter search tool to show that increased expression of OLA1 mRNA was significantly associated with shorter overall survival in lung cancer patients. By immunohistochemical analysis we discovered that levels of OLA1 protein in lung cancer tissues were positively correlated with TNM stage and lymph node metastasis, but negatively correlated with the epithelial-mesenchymal transition (EMT) marker E-cadherin. Knockdown of OLA1 in a lung adenocarcinoma cell line rendered the cells more resistant to TGF-ß-induced EMT and the accompanied repression of E-cadherin. Furthermore, our results demonstrated that OLA1 is a GSK3ß-interacting protein and inhibits GSK3ß activity by mediating its Ser9 phosphorylation. During EMT, OLA1 plays an important role in suppressing the GSK3ß-mediated degradation of Snail protein, which in turn promotes downregulation of E-cadherin. These data suggest that OLA1 contributes to EMT by modulating the GSK3ß/Snail/E-cadherin signaling, and its overexpression is associated with clinical progression and poor survival in lung cancer patients.

Aislamiento de Escherichia coli productora de toxina Shiga (STEC) en heces de ganado y detección de factores de virulencia asociados con su patogénesis / Isolation of Shiga toxin-producing Escherichia coli (STEC) in cattle stool and detection of virulence factors associated with its pathogenesis

Resumen Objetivo: Aislar STEC en las heces del ganado bovino en el municipio de Ulloa, Valle del Cauca y detectar factores de virulencia asociados con la patogénesis. Materiales y métodos: Se tomaron 21 muestras provenientes de bovinos, las cuales fueron tomadas directamente del recto del animal mediante hisopos. Las muestras se procesaron hasta obtener colonias puras a las cuales se les evaluó la presencia de los genes stx1, stx2, eae, saa y hlyA mediante PCR y posteriormente, se evaluó el efecto citotóxico de las muestras positivas sobre células Vero (ATCC-CCL-81.4). Resultados: De las 21 muestras de heces de bovinos,12 presentaron bacterias con uno o ambos genes stx. Se obtuvieron 106 aislamientos totales de STEC y se observaron diferencias en cuanto a la presencia y ausencia de los genes de virulencia evaluados en los aislamientos de cada bovino, obteniendo cinco combinaciones de genes. 48 aislamientos presentaron únicamente el gen stx2 y 58 presentaron tanto el gen stx1 como el gen stx2; de los 106 aislamientos, se detectaron 44 con el gen hlyA y 57 con el gen saa. Conclusiones: Todos los sobrenadantes de STEC analizados mostraron actividad citotóxica sobre las células Vero, mientras que en ausencia de STEC las células formaron monocapa después de 48 h de incubación. Este trabajo es el primer reporte en Colombia que aporta información sobre la presencia de STEC en el ganado bovino, la presencia de factores de virulencia y el potencial efecto citotóxico que poseen estas cepas nativas.

Abstract Objective: To isolate STEC in stool samples from cattle in Ulloa, Valle del Cauca, and to detect virulence factors associated with its pathogenesis. Materials and methods: We took 21 samples from cattle, which were taken directly from the rectum of the animal using swabs. The samples were processed until obtaining pure colonies and evaluated for the presence of the stx1, stx2, eae, saa and hlyA genes by PCR. Afterward, the cytotoxic effect of positive samples were evaluated on Vero cells (ATCC-CCL- 81.4). Results: We observed that from the 21 stools samples, 12 presented bacteria with one or both stx genes. A total of 106 isolates of STEC were obtained and differences among each other were observed regarding the presence and absence of the virulence genes, obtaining five combinations of genes. We found that 48 isolates presented only stx2 gene and 58 presented both the stx1 and stx2 gene. Regarding the other virulence genes, the hlyA gene was detected in 44 isolates and the saa gene was detected in 57 isolates. Conclusions: All the STEC supernatants showed cytotoxic activity on Vero cells, while in its absence the cells formed monolayer after 48 h of incubation. This work is the first report in Colombia that provides information about the presence of STEC in stool cattle, virulence genes and its potential cytotoxic effect in native strains.

OLA1 regulates protein synthesis and integrated stress response by inhibiting eIF2 ternary complex formation.

Translation is a fundamental cellular process, and its dysregulation can contribute to human diseases such as cancer. During translation initiation the eukaryotic initiation factor 2 (eIF2) forms a ternary complex (TC) with GTP and the initiator methionyl-tRNA (tRNAi), mediating ribosomal recruitment of tRNAi. Limiting TC availability is a central mechanism for triggering the integrated stress response (ISR), which suppresses global translation in response to various cellular stresses, but induces specific proteins such as ATF4. This study shows that OLA1, a member of the ancient Obg family of GTPases, is an eIF2-regulatory protein that inhibits protein synthesis and promotes ISR by binding eIF2, hydrolyzing GTP, and interfering with TC formation. OLA1 thus represents a novel mechanism of translational control affecting de novo TC formation, different from the traditional model in which phosphorylation of eIF2α blocks the regeneration of TC. Depletion of OLA1 caused a hypoactive ISR and greater survival in stressed cells. In vivo, OLA1-knockdown rendered cancer cells deficient in ISR and the downstream proapoptotic effector, CHOP, promoting tumor growth and metastasis. Our work suggests that OLA1 is a novel translational GTPase and plays a suppressive role in translation and cell survival, as well as cancer growth and progression.

Essential role of Nrf2 in protection against hydroquinone- and benzoquinone-induced cytotoxicity.

Benzene is a well-established human carcinogen. Benzene metabolites hydroquinone (HQ) and benzoquinone (BQ) are highly reactive molecules capable of producing reactive oxygen species and causing oxidative stress. In this study, we investigated the role of the Nrf2, a key nuclear transcription factor that regulates antioxidant response element (ARE)-containing genes, in defense against HQ- and BQ-induced cytotoxicity in cultured human lung epithelial cells (Beas-2B). When the cells were exposed to HQ or BQ the activity of an ARE reporter was induced in a dose-dependent manner, meanwhile Nrf2 protein levels were elevated and accumulated in the nucleus. Increased expression of well-known Nrf2-dependent proteins including NQO1, GCLM, GSS and HMOX was also observed in the HQ/BQ-treated cells. Moreover, transient overexpression of Nrf2 conferred protection against HQ- and BQ-induced cell death, whereas knockdown of Nrf2 by small interfering RNA resulted in increased apoptosis. We also found that the increased susceptibility of Nrf2-knockdown cells to HQ and BQ was associated with reduced glutathione levels and loss of inducibility of ARE-driven genes, suggesting that deficiency of Nrf2 impairs cellular redox capacity to counteract oxidative damage. Altogether, these results suggest that Nrf2-ARE pathway is essential for protection against HQ- and BQ-induced toxicity.

Cancer treatment using an optically inert Rose Bengal derivative combined with pulsed focused ultrasound.

Pulsed high intensity focused ultrasound (HIFU) has been combined with a photo-insensitive Rose Bengal derivative (RB2) to provide a synergistic cytotoxicity requiring the presence of both ultrasonic cavitation and drug. In vitro tests have shown that a short treatment (less than 30 s) of pulsed HIFU with peak negative pressure >7 MPa (~27 W acoustic power at 1.4 MHz) destroys >95% of breast cancer cells MDA-MB-231 in suspension with >10 µM of the compound. Neither the pulsed HIFU nor the RB2 compound was found to have any significant impact on the viability of the cells when used alone. Introducing an antioxidant (N-acetylcysteine) reduced the effectiveness of the treatment. In vivo tests using these same cells growing as a xenograft in nu/nu mice were also done. An ultrasound contrast agent (Optison) and lower frequency (1.0 MHz) was used to help initiate cavitation at the tumor site. We were able to demonstrate tumor regression with cavitation alone, however, addition of RB2 compound injected i.v. yielded a substantial synergistic improvement.

Effects of atmospheric pollutants on the Nrf2 survival pathway.

BACKGROUND, AIM, AND SCOPE: Atmospheric pollution is a worldwide problem. Exposure to atmospheric pollutants causes toxic cellular effects. One of the mechanisms of toxicity by these pollutants is the promotion of oxidative stress. Several signaling pathways control cellular redox homeostasis. In this respect, nuclear factor erythroid 2-related factor 2 (Nrf2) is a crucial transcription factor in the cell's response to oxidative stress. MAIN FEATURES: In cellular animal models, exposure to atmospheric pollutants activates Nrf2, attenuating its toxic and even its carcinogenic effects. Therefore, we have reviewed the scientific literature in order to indicate that air pollutants, such as particulate matter, polycyclic aromatic hydrocarbons, and gaseous matter, are Nrf2 pathway inductors, triggering self-defense through the establishment of proinflammatory and antioxidant responses. RESULTS AND DISCUSSION: Exposure to reactive molecules as atmospheric pollutants causes the activation of Nrf2 and the subsequent regulation of the expression of cytoprotective and detoxifying enzymes, as well as antioxidants. Moreover, induction of Nrf2 prior to exposure reduces the harmful effects of pollutants. The present article discusses the protective role of the Nrf2 pathway against different atmospheric pollutant insults. CONCLUSIONS: Nrf2 regulates the expression of numerous cytoprotective genes that function to detoxify reactive species produced during atmospheric pollutant metabolic reactions. From the papers highlighted in this review, we conclude that Nrf2 has an important role in the defense against atmospheric pollutant-induced toxicity. PERSPECTIVES: Further studies are needed to understand the signaling events that turn on the system in response to atmospheric pollutant stress. This could allow for the possibility of targeting the pathway for prevention benefits in the near future.

Knockdown of OLA1, a regulator of oxidative stress response, inhibits motility and invasion of breast cancer cells.

To explore the role of a novel Obg-like ATPase 1 (OLA1) in cancer metastasis, small interference RNA (siRNA) was used to knockdown the protein, and the cells were subjected to in vitro cell migration and invasion assays. Knockdown of OLA1 significantly inhibited cell migration and invasion in breast cancer cell line MDA-MB-231. The knockdown caused no changes in cell growth but affected ROS production. In wound-healing assays, decreased ROS in OLA1-knockdown cells were in situ associated with the cells' decreased motile morphology. Further, treatment of N-acetylcysteine, a general ROS scavenger, blunted the motility and invasiveness of MDA-MB-231 cells, similar to the effect of OLA1-knockdown. These results suggest that knockdown of OLA1 inhibits breast cancer cell migration and invasion through a mechanism that involves the modulation of intracellular ROS levels.

Coordination chemistry based approach to lipophilic inhibitors of 1-deoxy-D-xylulose-5-phosphate reductoisomerase.

1-Deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) in the non-mevalonate pathway found in most bacteria is a validated anti-infective drug target. Fosmidomycin, a potent DXR inhibitor, is active against Gram-negative bacteria. A coordination chemistry and structure based approach was used to discover a novel, lipophilic DXR inhibitor with an IC(50) of 1.4 microM. It exhibited a broad spectrum of activity against Gram-negative and -positive bacteria with minimal inhibition concentrations of 20-100 microM (or 3.7-19 microg/mL).