Histologic Evidence of Epithelial-Mesenchymal Transition and Autophagy in Human Fetal Membranes.

Preterm, prelabor rupture of the human fetal membranes (pPROM) is involved in 40% of spontaneous preterm births worldwide. Cellular-level disturbances and inflammation are effectors of membrane degradation, weakening, and rupture. Maternal risk factors induce oxidative stress (OS), senescence, and senescence-associated inflammation of the fetal membranes as reported mechanisms related to pPROM. Inflammation can also arise in fetal membrane cells (amnion/chorion) due to OS-induced autophagy and epithelial-mesenchymal transition (EMT). Autophagy, EMT, and their correlation in pPROM, along with OS-induced autophagy-related changes in amnion and chorion cells in vitro, were investigated. Immunocytochemistry staining of cytokeratin-18 (epithelial marker)/vimentin (mesenchymal marker) and proautophagy-inducing factor LC3B were performed in fetal membranes from pPROM, term not in labor, and term labor. Ultrastructural changes associated with autophagy were verified by transmission electron microscopy of the fetal membranes and in cells exposed to cigarette smoke extract (an OS inducer). EMT and LC3B staining was compared in the chorion from pPROM versus term not in labor. Transmission electron microscopy confirmed autophagosome formation in pPROM amnion and chorion. In cell culture, autophagosomes were formed in the amnion with OS treatment, while autophagosomes were accumulated in both cell types with autophagy inhibition. This study documents the association between pPROMs and amniochorion autophagy and EMT, and supports a role for OS in inducing dysfunctional cells that increase inflammation, predisposing membranes to rupture.

A Microphysiological Device to Model the Choriodecidual Interface Immune Status during Pregnancy.

During human pregnancy the chorion (fetal) lines decidua (maternal) creating the feto-maternal interface. Despite their proximity, resident decidual immune cells remain quiescent during gestation and do not invade the chorion. Infection and infiltration of activated immune cells toward the chorion are often associated with preterm birth. However, the mechanisms that maintain choriodecidual immune homeostasis or compromise immune barrier functions remain unclear. To understand these processes, a two-chamber microphysiological system (MPS) was created to model the human choriodecidual immune interface under normal and infectious conditions in vitro. This MPS has outer (fetal chorion trophoblast cells) and inner chambers (maternal decidual + CD45+ cells [70:30 ratio]) connected by microchannels. Decidual cells were treated with LPS to mimic maternal infection, followed by immunostaining for HLA-DR and HLA-G, immune panel screening by imaging cytometry by time of flight, and immune regulatory factors IL-8 and IL-10, soluble HLA-G, and progesterone (ELISA). LPS induced a proinflammatory phenotype in the decidua characterized by a decrease in HLA-DR and an increase in IL-8 compared with controls. LPS treatment increased the influx of immune cells into the chorion, indicative of chorionitis. Cytometry by time of flight characterized immune cells in both chambers as active NK cells and neutrophils, with a decrease in the abundance of nonproinflammatory cytokine-producing NK cells and T cells. Conversely, chorion cells increased progesterone and soluble HLA-G production while maintaining HLA-G expression. These results highlight the utility of MPS to model choriodecidual immune cell infiltration and determine the complex maternal-fetal crosstalk to regulate immune balance during infection.

Development of oxidative stress-associated disease models using feto-maternal interface organ-on-a-chip.

Oxidative stress (OS) and inflammation arising from cellular derangements at the fetal membrane-decidual interface (feto-maternal interface [FMi]) is a major antecedent to preterm birth (PTB). However, it is impractical to study OS-associated FMi disease state during human pregnancy, and thus it is difficult to develop strategies to reduce the incidences of PTB. A microfluidic organ-on-chip model (FMi-OOC) that mimics the in vivo structure and functions of FMi in vitro was developed to address this challenge. The FMi-OOC contained fetal (amnion epithelial, mesenchymal, and chorion) and maternal (decidua) cells cultured in four compartments interconnected by arrays of microchannels to allow independent but interconnected co-cultivation. Using this model, we tested the effects of OS and inflammation on both fetal (fetal → maternal) and maternal (maternal → fetal) sides of the FMi and determined their differential impact on PTB-associated pathways. OS was induced using cigarette smoke extract (CSE) exposure. The impacts of OS were assessed by measuring cell viability, disruption of immune homeostasis, epithelial-to-mesenchymal transition (EMT), development of senescence, and inflammation. CSE propagated (LC/MS-MS analysis for nicotine) over a 72-hour period from the maternal to fetal side, or vice versa. However, they caused two distinct pathological effects on the maternal and fetal cells. Specifically, fetal OS induced cellular pathologies and inflammation, whereas maternal OS caused immune intolerance. The pronounced impact produced by the fetus supports the hypothesis that fetal inflammatory response is a mechanistic trigger for parturition. The FMi disease-associated changes identified in the FMi-OOC suggest the unique capability of this in vitro model in testing in utero conditions.

A dynamic flow fetal membrane organ-on-a-chip system for modeling the effects of amniotic fluid motion.

Fetal membrane (amniochorion), the innermost lining of the intrauterine cavity, surround the fetus and enclose amniotic fluid. Unlike unidirectional blood flow, amniotic fluid subtly rocks back and forth, and thus, the innermost amnion epithelial cells are continuously exposed to low levels of shear stress from fluid undulation. Here, we tested the impact of fluid motion on amnion epithelial cells (AECs) as a bearer of force impact and their potential vulnerability to cytopathologic changes that can destabilize fetal membrane functions. A previously developed amnion membrane (AM) organ-on-chip (OOC) was utilized but with dynamic flow to culture human fetal amnion membrane cells. The applied flow was modulated to perfuse culture media back and forth for 48 h to mimic fluid motion. A static culture condition was used as a negative control, and oxidative stress (OS) condition was used as a positive control representing pathophysiological changes. The impacts of fluidic motion were evaluated by measuring cell viability, cellular transition, and inflammation. Additionally, scanning electron microscopy (SEM) imaging was performed to observe microvilli formation. The results show that regardless of the applied flow rate, AECs and AMCs maintained their viability, morphology, innate meta-state, and low production of pro-inflammatory cytokines. E-cadherin expression and microvilli formation in the AECs were upregulated in a flow rate-dependent fashion; however, this did not impact cellular morphology or cellular transition or inflammation. OS treatment induced a mesenchymal morphology, significantly higher vimentin to cytokeratin 18 (CK-18) ratio, and pro-inflammatory cytokine production in AECs, whereas AMCs did not respond in any significant manner. Fluid motion and shear stress, if any, did not impact AEC cell function and did not cause inflammation. Thus, when using an amnion membrane OOC model, the inclusion of a dynamic flow environment is not necessary to mimic in utero physiologic cellular conditions of an amnion membrane.

Modeling ascending Ureaplasma parvum infection through the female reproductive tract using vagina-cervix-decidua-organ-on-a-chip and feto-maternal interface-organ-on-a-chip.

Genital mycoplasmas can break the cervical barrier and cause intraamniotic infection and preterm birth. This study developed a six-chamber vagina-cervix-decidua-organ-on-a-chip (VCD-OOC) that recapitulates the female reproductive tract during pregnancy with culture chambers populated by vaginal epithelial cells, cervical epithelial and stromal cells, and decidual cells. Cells cultured in VCD-OOC were characterized by morphology and immunostaining for cell-specific markers. We transferred the media from the decidual cell chamber of the VCD-OOC to decidual cell chamber in feto-maternal interface organ-on-a-chip (FMi-OOC), which contains the fetal membrane layers. An ascending Ureaplasma parvum infection was created in VCD-OOC. U. parvum was monitored for 48 h post-infection with their cytotoxicity (LDH assay) and inflammatory effects (multiplex cytokine assay) in the cells tested. An ascending U. parvum infection model of PTB was developed using CD-1 mice. The cell morphology and expression of cell-specific markers in the VCD-OOC mimicked those seen in lower genital tract tissues. U. parvum reached the cervical epithelial cells and decidua within 48 h and did not cause cell death in VCD-OOC or FMi-OOC cells. U. parvum infection promoted minimal inflammation, while the combination of U. parvum and LPS promoted massive inflammation in the VCD-OOC and FMi-OOC cells. In the animal model, U. parvum vaginal inoculation of low-dose U. parvum did not result in PTB, and even a high dose had only some effects on PTB (20%). However, intra-amniotic injection of U. parvum resulted in 67% PTB. We report the colonization of U. parvum in various cell types; however, inconsistent, and low-grade inflammation across multiple cell types suggests poor immunogenicity induced by U. parvum.

Ureaplasma parvum infection induces inflammatory changes in vaginal epithelial cells independent of sialidase.

BACKGROUND: Ureaplasma, a genus of the order Mycoplasmatales and commonly grouped with Mycoplasma as genital mycoplasma is one of the most common microbes isolated from women with infection/inflammation-associated preterm labor (PTL). Mycoplasma spp. produce sialidase that cleaves sialic acid from glycans of vaginal mucous membranes and facilitates adherence and invasion of the epithelium by pathobionts, and dysregulated immune response. However, whether Ureaplasma species can induce the production of sialidase is yet to be demonstrated. We examined U. parvum-infected vaginal epithelial cells (VECs) for the production of sialidase and pro-inflammatory cytokines. METHODS: Immortalized VECs were cultured in appropriate media and treated with U. parvum in a concentration of 1 × 105 DNA copies/ml. After 24 h of treatment, cells and media were harvested. To confirm infection and cell uptake, immunocytochemistry for multi-banded antigen (MBA) was performed. Pro-inflammatory cytokine production and protein analysis for sialidase confirmed pro-labor pathways. RESULTS: Infection of VECs was confirmed by the presence of intracellular MBA. Western blot analysis showed no significant increase in sialidase expression from U. parvum-treated VECs compared to uninfected cells. However, U. parvum infection induced 2-3-fold increased production of GM-CSF (p = 0.03), IL-6 (p = 0.01), and IL-8 (p = 0.01) in VECs compared to controls. CONCLUSION: U. parvum infection of VECs induced inflammatory imbalance associated with vaginal dysbiosis but did not alter sialidase expression at the cellular level. These data suggest that U. parvum's pathogenic effect could be propagated by locally produced pro-inflammatory cytokines and, unlike other genital mycoplasmas, may be independent of sialidase.

Stress signaler p38 mitogen-activated kinase activation: a cause for concern?

Oxidative stress (OS) induced activation of p38 mitogen-activated kinase (MAPK) and cell fate from p38 signaling was tested using the human fetal membrane's amnion epithelial cells (AEC). We created p38 KO AEC using the CRISPR/Cas9 approach and tested cell fate in response to OS on an AEC-free fetal membrane extracellular matrix (ECM). Screening using image CyTOF indicated OS causing epithelial-mesenchymal transition (EMT). Further testing revealed p38 deficiency prevented AEC senescence, EMT, cell migration, and inflammation. To functionally validate in vitro findings, fetal membrane-specific conditional KO (cKO) mice were developed by injecting Cre-recombinase encoded exosomes intra-amniotically into p38αloxP/loxP mice. Amnion membranes from p38 cKO mice had reduced senescence, EMT, and increased anti-inflammatory IL-10 compared with WT animals. Our study suggested that overwhelming activation of p38 in response to OS inducing risk exposures can have an adverse impact on cells, cause cell invasion, inflammation, and ECM degradation detrimental to tissue homeostasis.

Organ-on-chip of the cervical epithelial layer: A platform to study normal and pathological cellular remodeling of the cervix.

Damage to the cervical epithelial layer due to infection and inflammation is associated with preterm birth. However, the individual and/or collective roles of cervical epithelial layers in maintaining cervical integrity remain unclear during infection/inflammation. To determine the intercellular interactions, we developed an organ-on-chip of the cervical epithelial layer (CE-OOC) composed of two co-culture chambers connected by microchannels, recapitulating the ectocervical and endocervical epithelial layers. Further, we tested the interactions between cells from each distinct region and their contributions in maintaining cervical integrity in response to LPS and TNFα stimulations. The co-culture of ectocervical and endocervical cells facilitated cellular migration of both epithelial cells inside the microchannels. Compared to untreated controls, both LPS and TNFα increased apoptosis, necrosis, and senescence as well as increased pro-inflammatory cytokine productions by cervical epithelial cells. In summary, the CE-OOC established an in vitro model that can recapitulate the ectocervical and the endocervical epithelial regions of the cervix. The established CE-OOC may become a powerful tool in obstetrics and gynecology research such as in studying cervical remodeling during pregnancy and parturition and the dynamics of cervical epithelial cells in benign and malignant pathology in the cervix.

Progesterone receptor membrane components: key regulators of fetal membrane integrity.

Pro-pregnancy hormone progesterone (P4) helps to maintain a quiescent status of uterine tissues during gestation. However, P4's functional role in maintaining fetal membrane (amniochorion) integrity remains unclear. P4 functions through its membrane receptors (progesterone receptor membrane components (PGRMCs)) as fetal membrane cells lack nuclear receptors. This study screened the differential expression of PGRMCs in the fetal membranes and tested P4-PGRMC interactions under normal and oxidative stress (OS) conditions expected that can disrupt P4-PGRMC interactions impacting fetal membrane stability resulting in parturition. Human fetal membranes were collected from term and preterm deliveries (N = 5). Immunohistochemistry and western blot localized and determined differential expression of P4 receptors. Primary amnion epithelial, mesenchymal (AMCs), and chorion cell were treated with P4 alone or co-treated (P4 + OS induced by cigarette smoke extract (CSE)). Proximity ligation assay (PLA) documented P4-receptor binding, whereas P4 enzyme-linked immunosorbent assay documented culture supernatant levels. Immunohistology confirmed lack of nuclear progesterone receptors; however, confirmed expressions of PGRMC 1 and 2. Term labor (P = 0.01) and preterm rupture (P = 0.01) are associated with significant downregulation of PGRMC2. OS-induced differential downregulation of PGRMCs in both amnion and chorion cells (all P < 0.05) and downregulates P4 release (AMCs; P = 0.01). The PLA showed preferential receptor-ligand binding in amnion and chorion cells. Co-treatment of P4 + CSE did not reverse CSE-induced effects. In conclusion, P4-PGRMCs interaction maintains fetal membranes' functional integrity throughout pregnancy. Increased OS reduces endogenous P4 production and cell type-dependent downregulation of PGRMCs. These changes can lead to fetal membrane-specific "functional progesterone withdrawal," contributing to the dysfunctional fetal membrane status seen at term and preterm conditions.

Birds, blooms, and evolving diversity.

In this Open Highlight, Senior Editor Lauren Richardson features exciting new Open Access research into how species evolve their characteristic traits.

Inflammation, but not infection, induces EMT in human amnion epithelial cells.

A non-reversible state of epithelial to mesenchymal transition (EMT) at term accumulates proinflammatory mesenchymal cells and predisposes fetal membrane to weakening prior to delivery at term. We investigated the induction of EMT in amnion epithelial cells (AEC) in response to inflammation and infection associated with spontaneous preterm birth (SPTB). For this, membranes from SPTB were screened for EMT markers. Primary AEC in culture were treated with TNF-α (10 and 50 ng/mL) and LPS (50 and 100 ng/mL) for 72 h. Cell shape index (SI) was determined based on morphological shift (microscopy followed by ImageJ software analysis). Immunocytochemistry and Western blot assessed changes in epithelial markers (cytokeratin-18 and E-cadherin) and mesenchymal markers (vimentin and N-cadherin). Involvement of transforming growth factor beta (TGF-ß) in EMT induction and EMT associated inflammation was tested using specific markers (Western blot) and by measuring MMP9 (ELISA), respectively. We report that PTB is associated with fetal membrane EMT. TNF-α produced dose- and time-dependent induction of EMT; within 24 h by 50 ng/mL and after 72 h by 10 ng/mL. AEC showed mesenchymal morphology, lower E-cadherin, higher vimentin and N-cadherin and higher MMP9 compared to control. TNF-α-induced EMT was not associated with canonical TGF-ß pathway. LPS, regardless of dose or time, did not induce EMT in AEC. We conclude that PTB with intact membranes is associated with EMT. Our data suggest that inflammation, but not infection, is associated with non-canonical activation of EMT and inflammation that can predispose membrane to undergo weakening.

Amnion membrane organ-on-chip: an innovative approach to study cellular interactions.

The amnion membrane that lines the human intrauterine cavity is composed of amnion epithelial cells (AECs) connected to an extracellular matrix containing amnion mesenchymal cells (AMCs) through a basement membrane. Cellular interactions and transitions are mechanisms that facilitate membrane remodeling to maintain its integrity. Dysregulation of cellular remodeling, primarily mediated by oxidative stress (OS), is often associated with preterm birth. However, the mechanisms that maintain membrane homeostasis remain unclear. To understand these mechanisms, we developed an amnion membrane organ-on-chip (AM-OOC) and tested the interactive and transition properties of primary human AECs and AMCs under normal and OS conditions. AM-OOC contained 2 chambers connected by type IV collagen-coated microchannels, allowing independent culture conditions that permitted cellular migration and interactions. Cells grown either independently or coculture were exposed to OS inducing cigarette smoke extract, antioxidant N-acetyl-l-cysteine (NAC), or both. When grown independently, AECs transitioned to AMCs and migrated, whereas AMCs migrated without transition. OS caused AECs' transition but prevented migration, whereas AMCs' migration was unhindered. Coculture of cells facilitated transition, migration, and eventual integration in the contiguous population. OS cotreatment in both chambers facilitated AECs' transition, prevented migration, and increased inflammation, a process that was prevented by NAC. AM-OOC recapitulated cellular mechanisms observed in utero and enabled experimental manipulation of cells to determine their roles during pregnancy and parturition.-Richardson, L., Jeong, S., Kim, S., Han, A., Menon, R. Amnion membrane organ-on-chip: an innovative approach to study cellular interactions.

Dexamethasone induces primary amnion epithelial cell senescence through telomere-P21 associated pathway†.

Dexamethasone (Dex), a corticosteroid hormone, is used during the perinatal period to help fetal lung and other organ development. Conversely, Dex-induced cell proliferation has been associated with accelerated aging. Using primary amnion epithelial cells (AECs) from term, not in labor, fetal membranes, we tested the effects of Dex on cell proliferation, senescence, and inflammation. Primary AECs treated with Dex (100 and 200 nM) for 48 h were tested for cell viability (crystal violet dye exclusion), cell cycle progression and/or type of cell death (flow cytometry), expression patterns of steroid receptors (glucocorticoid receptor, progesterone receptor membrane component 1&2), inflammatory mediators (IL-6 and IL-8), and telomere length (quantitative RT-PCR). Mechanistic mediators of senescence (p38MAPK and p21) were determined by western blot analysis. Dex treatment did not induce AEC proliferation, cell cycle, influence viability, or morphology. However, Dex caused dependent telomere length reduction and p38MAPK-independent but p21-dependent (confirmed by treatment with p21 inhibitor UC2288). Senescence was not associated with an increase in inflammatory mediators, which is often associated with senescence. Co-treatment with RU486 produced DNA damage, cell cycle arrest, and cellular necrosis with an increase in inflammatory mediators. The effect of Dex was devoid of changes to steroid receptors, whereas RU486 increased GR expression. Dex treatment of AECs produced nonreplicative and noninflammatory senescence. Extensive use of Dex during the perinatal period may lead to cellular senescence, contributing to cellular aging associated pathologies during the perinatal and neonatal periods.

Oxidative stress-induced downregulation of glycogen synthase kinase 3 beta in fetal membranes promotes cellular senescence†.

OBJECTIVE: Oxidative stress (OS)-induced stress signaler p38 mitogen-activated protein kinase (p38MAPK) activation and fetal membrane senescence are associated with parturition. This study determined changes in glycogen synthase kinase 3 beta (GSK3ß) and its regulation by p38MAPK in effecting senescence to further delineate the molecular mechanism involved in senescence. METHODS: Primary human amnion epithelial cells and amnion mesenchymal cells were treated with cigarette smoke extract (CSE, OS inducer). Expression of total and phosphorylated GSK3ß and p38MAPK, and that of GSK3ß's downstream targets: beta-catenin (ß-Cat) and nuclear factor erythroid 2-related factor 2 (Nrf2) (western blot analysis), cell cycle regulation and senescence (flow cytometry) were determined. The specificity of GSK3ß and p38MAPK's mechanistic role was tested by co-treating cells with their respective inhibitors, CHIR99021 and SB203580. Exosomal secretion of ß-Cat from OS-induced cells was confirmed by immunofluorescence confocal microscopy and western blot. RESULTS: OS induced by CSE resulted in phosphorylation of GSK3ß (inactivation) and p38MAPK (activation) that was associated with cell cycle arrest and senescence. Inhibitors to GSK3ß and p38MAPK verified their roles. Glycogen synthase kinase 3 beta inactivation was associated with nuclear translocation of antioxidant Nrf2 and exosomal secretion of ß-Cat. CONCLUSIONS: OS-induced P-p38MAPK activation is associated with functional downregulation of GSK3ß and arrest of cell cycle progression and senescence of amnion cells. Lack of nuclear translocation of ß-Cat and its excretion via exosomes further supports the postulation that GSK3ß down-regulation by p38MAPK may stop cell proliferation preceding cell senescence. A better understanding of molecular mechanisms of senescence will help develop therapeutic strategies to prevent preterm birth.

Proliferative, Migratory, and Transition Properties Reveal Metastate of Human Amnion Cells.

Amnion epithelial cell (AEC) shedding causes microfractures in human placental membranes during gestation. However, microfractures are healed to maintain membrane integrity. To better understand the cellular mechanisms of healing and tissue remodeling, scratch assays were performed using primary AECs derived from normal term not in labor membranes. AECs were grown under different conditions: i) normal cultures (control), ii) oxidative stress (OS) induction by cigarette smoke extract (CSE), iii) co-treatment of CSE and antioxidant N-acetyl-l-cysteine, and iv) treatment with amniotic fluid (AF). Cell migration time and distance, changes in intermediate filament (cytokeratin-18 and vimentin) expressions, and cellular senescence were determined. Control AECs in culture exhibited a metastate with the expression of both cytokeratin-18 and vimentin. During healing, AECs proliferated, migrated, and transitioned from epithelial to mesenchymal phenotype with increased vimentin. Wound healing was associated with mesenchymal to epithelial transition (MET). CSE-induced OS and senescence prevented wound healing in which cells sustained mesenchymal state. N-acetyl-l-cysteine reversed CSE's effect to aid wound closure through MET. AF accelerated cellular transitions and healing. Our data suggest that AECs undergo epithelial to mesenchymal transition during proliferation and migration and MET at the injury site to promote healing. AF accelerated whereas OS diminished cellular transitions and healing. OS-inducing pregnancy risk factors may diminish remodeling capacity contributing to membrane dysfunction, leading to preterm birth.

Understanding Disease Tolerance and Resilience.

Our first ever Open Highlights explores recent Open Access research into the complex relationship between host and pathogen during the course of an infection, and the factors that determine its eventual outcome.

Disorder targets misorder in nuclear quality control degradation: a disordered ubiquitin ligase directly recognizes its misfolded substrates.

Protein quality control (PQC) degradation systems protect the cell from the toxic accumulation of misfolded proteins. Because any protein can become misfolded, these systems must be able to distinguish abnormal proteins from normal ones, yet be capable of recognizing the wide variety of distinctly shaped misfolded proteins they are likely to encounter. How individual PQC degradation systems accomplish this remains an open question. Here we show that the yeast nuclear PQC ubiquitin ligase San1 directly recognizes its misfolded substrates via intrinsically disordered N- and C-terminal domains. These disordered domains are punctuated with small segments of order and high sequence conservation that serve as substrate-recognition sites San1 uses to target its different substrates. We propose that these substrate-recognition sites, interspersed among flexible, disordered regions, provide San1 an inherent plasticity which allows it to bind its many, differently shaped misfolded substrates.

Oxidative stress-induced TGF-beta/TAB1-mediated p38MAPK activation in human amnion epithelial cells.

Term and preterm parturition are associated with oxidative stress (OS)-induced p38 mitogen-activated protein kinase (p38MAPK)-mediated fetal tissue (amniochorion) senescence. p38MAPK activation is a complex cell- and stimulant-dependent process. Two independent pathways of OS-induced p38MAPK activation were investigated in amnion epithelial cells (AECs) in response to cigarette smoke extract (CSE: a validated OS inducer in fetal cells): (1) the OS-mediated oxidation of apoptosis signal-regulating kinase (ASK)-1 bound Thioredoxin (Trx[SH]2) dissociates this complex, creating free and activated ASK1-signalosome and (2) transforming growth factor-mediated activation of (TGF)-beta-activated kinase (TAK)1 and TGF-beta-activated kinase 1-binding protein (TAB)1. AECs isolated from normal term, not-in-labor fetal membranes increased p38MAPK in response to CSE and downregulated it in response to antioxidant N-acetylcysteine. In AECs, both Trx and ASK1 were localized; however, they remained dissociated and not complexed, regardless of conditions. Silencing either ASK1 or its downstream effectors (MKK3/6) did not affect OS-induced p38MAPK activation. Conversely, OS increased TGF-beta's release from AECs and increased phosphorylation of both p38MAPK and TAB1. Silencing of TAB1, but not TAK1, prevented p38MAPK activation, which is indicative of TAB1-mediated autophosphorylation of p38MAPK, an activation mechanism seldom seen. OS-induced p38MAPK activation in AECs is ASK1-Trx signalosome-independent and is mediated by the TGF-beta pathway. This knowledge will help to design strategies to reduce p38MAPK activation-associated pregnancy risks.

Discovery and Characterization of Human Amniochorionic Membrane Microfractures.

This study obtained visual evidence of novel cellular and extracellular matrix-level structural alterations in term and preterm human fetal amniochorionic membranes. Amniochorions were collected from term cesarean (not in labor) or vaginal (labor) deliveries, preterm premature rupture of membranes, and spontaneous preterm birth. To determine the effect of oxidative stress on membranes at term or preterm labor, term not in labor samples in an organ explant culture in vitro were exposed to cigarette smoke extract. Tissues were imaged using multiphoton autofluorescence and second harmonic generation microscopy. Images were analyzed using ImageJ and IMARIS software. Three-dimensional microscopic analysis of membranes revealed microfractures that were characterized by amnion cell puckering, basement membrane degradation, and tunnels that extended into the collagen matrix with migrating cells. Numbers of microfractures were similar at term regardless of labor status; however, morphometric measures (width and depth) were higher in term labor membranes. Oxidative stress induced higher numbers of microfractures in term not in labor membranes, with morphometry resembling that seen in term labor membranes. Preterm premature rupture of the membranes had the highest number of microfractures compared to membranes from term and other preterm births. Microfractures are structural alterations indicative of areas of tissue remodeling during gestation. Their increase at preterm and in response to oxidative stress may indicate failure to reseal, predisposing membranes to rupture.

From Friend to Foe: Toxicity Trade-Offs Govern Staphylococcus aureus Infection Severity.

The life-threatening human pathogen Staphylococcus aureus experiences an evolutionary tug-of-war between highly toxic strains, which are better able to transmit between hosts and less toxic strains which are better at infecting a single host. Read the Research Article.