Downregulation of krüppel-like factor 6 expression modulates extravillous trophoblast cell behavior by increasing reactive oxygen species.

INTRODUCTION: Placental extravillous trophoblasts play a crucial role in the establishment of a healthy pregnancy. Reactive oxygen species (ROS) may contribute to their differentiation and function as mediators in signaling processes or might cause oxidative stress resulting in trophoblast dysfunction. The krüppel-like transcription factor 6 (KLF6) regulates many genes involved in essential cell processes where ROS are also involved. However, whether KLF6 regulates ROS levels has not been previously investigated. MATERIALS AND METHODS: KLF6 was silenced by siRNAs in HTR8-SV/neo cells, an extravillous trophoblast model. Total and mitochondrial ROS levels, as well as mitochondrial membrane potential and apoptosis were analyzed by flow cytometry. The expression of genes and proteins of interest were analyzed by qRT-PCR and Western blot, respectively. Cell response to oxidative stress, proliferation, viability, morphology, and migration were evaluated. RESULTS: KLF6 downregulation led to an increase in ROS and NOX4 mRNA levels, accompanied by reduced cell proliferation and increased p21 protein expression. Catalase activity, 2-Cys peroxiredoxin protein levels, Nrf2 cytoplasmic localization and hemoxygenase 1 expression, as well as mitochondrial membrane potential and cell apoptosis were not altered suggesting that ROS increase is not associated with cellular damage. Instead, KLF6 silencing induced cytoskeleton modifications and increased cell migration in a ROS-dependent manner. DISCUSSION: Present data reveal a novel role of KLF6 on ROS balance and signaling demonstrating that KLF6 downregulation induces an increase in ROS levels that contribute to extravillous trophoblast cell migration.

Krüppel-like factor 6 participates in extravillous trophoblast cell differentiation and its expression is reduced in abnormally invasive placenta.

Trophoblast cell differentiation is of paramount importance for successful pregnancy. Krüppel-like factor 6 (KLF6), a transcription factor with diverse roles in cell physiology and tumor biology, is required for trophoblast differentiation through the syncytial pathway. Herein, we demonstrate that extravillous trophoblast (EVT) cell migration and mesenchymal phenotype are increased upon KLF6 downregulation or the expression of a deletion mutant lacking its transcriptional regulatory domain (KΔac). Raman spectroscopy revealed molecular modifications compatible with increased differentiation in cells stably expressing the KΔac mutant. Moreover, abnormally invasive placenta showed lower KLF6 immunostaining compared with the normal placenta. Thus, impaired KLF6 expression or function stimulates EVT migration and differentiation in vitro and may contribute to the physiopathology of the abnormally invasive placenta.

Krüppel-like factor 6 (KLF6) requires its amino terminal domain to promote villous trophoblast cell fusion.

INTRODUCTION: Villous cytotrophoblast (vCTB) cells fuse to generate and maintain the syncytiotrophoblast layer required for placental development and function. Krüppel-like factor 6 (KLF6) is a ubiquitous transcription factor with an N-terminal acidic transactivation domain and a C-terminal zinc finger DNA-binding domain. KLF6 is highly expressed in placenta, and it is required for proper placental development. We have demonstrated that KLF6 is necessary for cell fusion in human primary vCTBs, and in the BeWo cell line. MATERIALS AND METHODS: Full length KLF6 or a mutant lacking its N-terminal domain were expressed in BeWo cells or in primary vCTB cells isolated from human term placentas. Cell fusion, gene and protein expression, and cell proliferation were analyzed. Moreover, Raman spectroscopy and atomic force microscopy (AFM) were used to identify biochemical, topography, and elasticity cellular modifications. RESULTS: The increase in KLF6, but not the expression of its deleted mutant, is sufficient to trigger cell fusion and to raise the expression of ß-hCG, syncytin-1, the chaperone protein 78 regulated by glucose (GRP78), the ATP Binding Cassette Subfamily G Member 2 (ABCG2), and Galectin-1 (Gal-1), all molecules involved in vCTB differentiation. Raman and AFM analysis revealed that KLF6 reduces NADH level and increases cell Young's modulus. KLF6-induced differentiation correlates with p21 upregulation and decreased cell proliferation. Remarkable, p21 silencing reduces cell fusion triggered by KLF6 and the KLF6 mutant impairs syncytialization and decreases syncytin-1 and ß-hCG expression. DISCUSSION: KLF6 induces syncytialization through a mechanism that involves its regulatory transcriptional domain in a p21-dependent manner.

Role of the lipid transport protein StarD7 in mitochondrial dynamics.

Mitochondria are dynamic organelles crucial for cell function and survival implicated in oxidative energy production whose central functions are tightly controlled by lipids. StarD7 is a lipid transport protein involved in the phosphatidylcholine (PC) delivery to mitochondria. Previous studies have shown that StarD7 knockdown induces alterations in mitochondria and endoplasmic reticulum (ER) with a reduction in PC content, however whether StarD7 modulates mitochondrial dynamics remains unexplored. Here, we generated HTR-8/SVneo stable cells expressing the precursor StarD7.I and the mature processed StarD7.II isoforms. We demonstrated that StarD7.I overexpression altered mitochondrial morphology increasing its fragmentation, whereas no changes were observed in StarD7.II-overexpressing cells compared to the control (Ct) stable cells. StarD7.I (D7.I) stable cells were able to transport higher fluorescent PC analog to mitochondria than Ct cells, yield mitochondrial fusions, maintained the membrane potential, and produced lower levels of reactive oxygen species (ROS). Additionally, the expression of Dynamin Related Protein 1 (Drp1) and Mitofusin (Mfn2) proteins were increased, whereas the amount of Mitofusin 1 (Mfn1) decreased. Moreover, transfections with plasmids encoding Drp1-K38A, Drp1-S637D or Drp1-S637A mutants indicated that mitochondrial fragmentation in D7.I cells occurs in a fission-dependent manner via Drp1. In contrast, StarD7 silencing decreased Mfn1 and Mfn2 fusion proteins without modification of Drp1 protein level. These cells increased ROS levels and presented donut-shape mitochondria, indicative of metabolic stress. Altogether our findings provide novel evidence indicating that alterations in StarD7.I expression produce significant changes in mitochondrial morphology and dynamics.

Phenotypic and genotypic characterization of Salmonella spp. isolated from foods and clinical samples in Brazil

ABSTRACT Increasing antimicrobial resistance in Salmonella species has been a serious problem for public health worldwide. This study examines Salmonella spp. recovered from foods and clinical samples on serotype, antimicrobial resistance and PFGE genotypes. It identified 91 salmonellae, belonging to 31 different serotypes, from 36 isolates from food and 55 clinical samples. Salmonella Infantis (16.5%) and Salmonella Enteritidis (13.7%) are the most common among food isolates, whereas Salmonella Enteritidis (29.0%) and Salmonella Typhimurium (16.0%) mainly causes human salmonellosis. Antimicrobial susceptibility data showed that 63.0% of the isolates were fully susceptible to 12 antibiotics tested. Nalidixic acid showed high resistance rates, 32.7% and 25.0% of the clinical isolates and food, respectively. Three main PFGE types: A (Salmonella Enteritidis), B (Salmonella Infantis) and C (Salmonella Schwarzengrund) comprised isolates recovered from foods and clinical samples. Our results indicate that the clonal groups were both causing diseases and food contamination, emphasizing the need to maintain a system of surveillance for foodborne disease.

Population dynamics of Siderastrea stellata Verrill, 1868 from Rocas Atoll, RN: implications for predicted climate change impacts at the only South Atlantic atoll.

Coral reefs are one of the most vulnerable ecosystems to ocean warming and acidification, and it is important to determine the role of reef building species in this environment in order to obtain insight into their susceptibility to expected impacts of global changes. Aspects of the life history of a coral population, such as reproduction, growth and size-frequency can contribute to the production of models that are used to estimate impacts and potential recovery of the population, acting as a powerful tool for the conservation and management of those ecosystems. Here, we present the first evidence of Siderastrea stellata planulation, its early growth, population size-frequency distribution and growth rate of adult colonies in Rocas Atoll. Our results, together with the environmental protection policies and the absence of anthropogenic pressures, suggest that S. stellata population may have a good potential in the maintenance and recovery in the atoll. However, our results also indicate an impact on corals' recruitment, probably as a consequence of the positive temperature anomaly that occurred in 2010. Thus, despite the pristine status of Rocas Atoll, the preservation of its coral community seems to be threatened by current global changes, such as more frequent thermal stress events.