Group B streptococcus induces cellular senescence in human amnion epithelial cells through a partial interleukin-1-mediated mechanism.

Group B streptococcus (GBS) infection is a significant public health concern associated with adverse pregnancy complications and increased neonatal mortality and morbidity. However, the mechanisms underlying the impact of GBS on the fetal membrane, the first line of defense against pathogens, are not fully understood. Here, we propose that GBS induces senescence and inflammatory factors (IL-6 and IL-8) in the fetal membrane through interleukin-1 (IL-1). Utilizing the existing transcriptomic data on GBS-exposed human fetal membrane, we showed that GBS affects senescence-related pathways and genes. Next, we treated primary amnion epithelial cells with conditioned medium from the choriodecidual layer of human fetal membrane exposed to GBS (GBS collected choriodecidual [CD] conditioned medium) in the absence or presence of an IL-1 receptor antagonist (IL-1Ra). GBS CD conditioned medium significantly increased ß-galactosidase activity, IL-6 and IL-8 release from the amnion epithelial cells. Cotreatment with IL1Ra reduced GBS-induced ß-galactosidase activity and IL-6 and IL-8 secretion. Direct treatment with IL-1α or IL-1ß confirmed the role of IL-1 signaling in the regulation of senescence in the fetal membrane. We further showed that GBS CD conditioned medium and IL-1 decreased cell proliferation in amnion epithelial cells. In summary, for the first time, we demonstrate GBS-induced senescence in the fetal membrane and present evidence of IL-1 pathway signaling between the choriodecidua and amnion layer of fetal membrane in a paracrine manner. Further studies will be warranted to understand the pathogenesis of adverse pregnancy outcomes associated with GBS infection and develop therapeutic interventions to mitigate these complications.

Factors associated with successful median arcuate ligament release in an international, multi-institutional cohort.

OBJECTIVE: Prior research on median arcuate ligament syndrome has been limited to institutional case series, making the optimal approach to median arcuate ligament release (MALR) and resulting outcomes unclear. In the present study, we compared the outcomes of different approaches to MALR and determined the predictors of long-term treatment failure. METHODS: The Vascular Low Frequency Disease Consortium is an international, multi-institutional research consortium. Data on open, laparoscopic, and robotic MALR performed from 2000 to 2020 were gathered. The primary outcome was treatment failure, defined as no improvement in median arcuate ligament syndrome symptoms after MALR or symptom recurrence between MALR and the last clinical follow-up. RESULTS: For 516 patients treated at 24 institutions, open, laparoscopic, and robotic MALR had been performed in 227 (44.0%), 235 (45.5%), and 54 (10.5%) patients, respectively. Perioperative complications (ileus, cardiac, and wound complications; readmissions; unplanned procedures) occurred in 19.2% (open, 30.0%; laparoscopic, 8.9%; robotic, 18.5%; P < .001). The median follow-up was 1.59 years (interquartile range, 0.38-4.35 years). For the 488 patients with follow-up data available, 287 (58.8%) had had full relief, 119 (24.4%) had had partial relief, and 82 (16.8%) had derived no benefit from MALR. The 1- and 3-year freedom from treatment failure for the overall cohort was 63.8% (95% confidence interval [CI], 59.0%-68.3%) and 51.9% (95% CI, 46.1%-57.3%), respectively. The factors associated with an increased hazard of treatment failure on multivariable analysis included robotic MALR (hazard ratio [HR], 1.73; 95% CI, 1.16-2.59; P = .007), a history of gastroparesis (HR, 1.83; 95% CI, 1.09-3.09; P = .023), abdominal cancer (HR, 10.3; 95% CI, 3.06-34.6; P < .001), dysphagia and/or odynophagia (HR, 2.44; 95% CI, 1.27-4.69; P = .008), no relief from a celiac plexus block (HR, 2.18; 95% CI, 1.00-4.72; P = .049), and an increasing number of preoperative pain locations (HR, 1.12 per location; 95% CI, 1.00-1.25; P = .042). The factors associated with a lower hazard included increasing age (HR, 0.99 per increasing year; 95% CI, 0.98-1.0; P = .012) and an increasing number of preoperative diagnostic gastrointestinal studies (HR, 0.84 per study; 95% CI, 0.74-0.96; P = .012) Open and laparoscopic MALR resulted in similar long-term freedom from treatment failure. No radiographic parameters were associated with differences in treatment failure. CONCLUSIONS: No difference was found in long-term failure after open vs laparoscopic MALR; however, open release was associated with higher perioperative morbidity. These results support the use of a preoperative celiac plexus block to aid in patient selection. Operative candidates for MALR should be counseled regarding the factors associated with treatment failure and the relatively high overall rate of treatment failure.

Trichloroethylene Metabolite S-(1,2-Dichlorovinyl)-l-cysteine Stimulates Changes in Energy Metabolites and Amino Acids in the BeWo Human Placental Trophoblast Model during Syncytialization.

Syncytialization, the fusion of cytotrophoblasts into an epithelial barrier that constitutes the maternal-fetal interface, is a crucial event of placentation. This process is characterized by distinct changes to amino acid and energy metabolism. A metabolite of the industrial solvent trichloroethylene (TCE), S-(1,2-dichlorovinyl)-l-cysteine (DCVC), modifies energy metabolism and amino acid abundance in HTR-8/SVneo extravillous trophoblasts. In the current study, we investigated DCVC-induced changes to energy metabolism and amino acids during forskolin-stimulated syncytialization in BeWo cells, a human villous trophoblastic cell line that models syncytialization in vitro. BeWo cells were exposed to forskolin at 100 µM for 48 h to stimulate syncytialization. During syncytialization, BeWo cells were also treated with DCVC at 0 (control), 10, or 20 µM. Following treatment, the targeted metabolomics platform, "Tricarboxylic Acid Plus", was used to identify changes in energy metabolism and amino acids. DCVC treatment during syncytialization decreased oleic acid, aspartate, proline, uridine diphosphate (UDP), UDP-d-glucose, uridine monophosphate, and cytidine monophosphate relative to forskolin-only treatment controls, but did not increase any measured metabolite. Notable changes stimulated by syncytialization in the absence of DCVC included increased adenosine monophosphate and guanosine monophosphate, as well as decreased aspartate and glutamate. Pathway analysis revealed multiple pathways in amino acid and sugar metabolisms that were altered with forskolin-stimulated syncytialization alone and DCVC treatment during syncytialization. Analysis of ratios of metabolites within the pathways revealed that DCVC exposure during syncytialization changed metabolite ratios in the same or different direction compared to syncytialization alone. Building off our oleic acid findings, we found that extracellular matrix metalloproteinase-2, which is downstream in oleic acid signaling, underwent the same changes as oleic acid. Together, the metabolic changes stimulated by DCVC treatment during syncytialization suggest changes in energy metabolism and amino acid abundance as potential mechanisms by which DCVC could impact syncytialization and pregnancy.

Utility of Hook Sign in the Diagnosis of Median Arcuate Ligament Syndrome.

BACKGROUND: Median arcuate ligament syndrome (MALS) is a clinical syndrome caused by compression of the celiac artery by the median arcuate ligament that often manifests with nonspecific abdominal pain. Identification of this syndrome is often dependent on imaging of compression and upward bending of the celiac artery by lateral computed tomography angiography, the so-called "hook sign." The purpose of this study was to assess the relationship of radiologic characteristics of the celiac artery to clinically relevant MALS. METHODS: An institutional review board-approved retrospective chart review from 2,000 to 2,021 of 293 patients at a tertiary academic center diagnosed with celiac artery compression (CAC) was performed. Patient demographics and symptoms of 69 patients who were diagnosed with symptomatic MALS were compared to 224 patients without MALS (but with CAC) per electronic medical record review. Computed tomography angiography images were reviewed and the fold angle (FA) was measured. The presence of a hook sign (defined as a visual FA < 135°), as well as stenosis (defined as >50% of luminal narrowing on imaging) were recorded. Wilcoxon rank-sum test and Chi-squared test were used for comparative analysis. Logistic model was run to relate the presence of MALS with comorbidities and radiographic findings. RESULTS: Imaging was available in 59 patients (25 males, 34 females) and 157 patients (60 males, 97 females) with and without MALS, respectively. Patients with MALS were more likely to have a more severe FA (120.7 ± 33.6 vs. 134.8 ± 27.9, P = 0.002). Males with MALS were also more likely to have a more severe FA compared with males without MALS (111.1 ± 33.7 vs. 130.4 ± 30.4, P = 0.015). In patients with body mass index (BMI) >25, MALS patients also had narrower FA compared with patients without MALS (112.6 ± 30.5 vs. 131.7 ± 30.3, P = 0.001). The FA was negatively correlated with BMI in patients with CAC. The hook sign and stenosis were associated with diagnosis of MALS (59.3% vs. 28.7%, P < 0.001, and 75.7% vs. 45.2%, P < 0.001, respectively). In logistic regression, pain, stenosis, and a narrow FA were statistically significant predictors of the presence of MALS. CONCLUSIONS: The upward deflection of the celiac artery in patients with MALS is more severe compared with patients without MALS. Consistent with prior literature, this bending of the celiac artery is negatively correlated with BMI in patients with and without MALS. When demographic variables and comorbidities are considered, a narrow FA is a statistically significant predictor of MALS. Regardless of MALS diagnosis, a hook sign was associated with narrower FA. While demographics and imaging findings may inform MALS diagnosis, clinicians should not rely on a visual assessment of a hook sign but should quantitatively measure the anatomic bending angle of the celiac artery to assist with the diagnosis and understand the outcomes.

Elucidating the Role of the AFX2 Endograft in Endovascular Treatment of Aortic Pathology.

BACKGROUND: The purpose of this study is to elucidate the role of the AFX2 platform in the endovascular treatment of aortic pathology. METHODS: All procedures by a single surgeon resulting in implantation of a bifurcated unibody stent graft were reviewed retrospectively. Indications for selection of the AFX2 endograft in each case were evaluated. Aortic anatomy was determined via review of pre-operative computed tomography (CT) scans. Cumulative event probabilities for endoleak, reintervention, and mortality were estimated. Patient and procedural details were described using mean, standard deviation, medians, and interquartile range (IQR). Kaplan-Meier survival analysis estimated freedom from mortality and reintervention. Cumulative incidence probabilities were calculated as one minus the Kaplan-Meier estimator. RESULTS: Between March 2018 and December 2020, the author (NN) used 142 aortic endografts in 142 patients. Of these, 46 (32.4%) were AFX2 endografts and the remaining were modular bifurcated devices, predominantly Medtronic Endurant II and Terumo Treo. No AFX-Strata or AFX-Duraply devices were placed. Amongst the patients who received an AFX2, mean age was 71.3 +/- 9.8 years with 84.8% male. Median operative time was 116 (86-166) min, with contrast dose of 79 (41-120) milliliters and fluoroscopy time of 12 (8.6-18) min. Overall, 78.3% (n = 36) of AFX2 devices were placed in aortas with maximum true lumen diameter <5.0 cm. Median postoperative follow-up was 1.7 years (IQR 1.0-2.4 years), with a maximum follow-up of 3.6 years. There was 1 patient lost to follow-up at 5 months. The 2-year incidence of type II endoleak, reintervention, and all-cause mortality was 12.7% (95% confidence interval CI, 0-29.6%), 2.2% (95% CI, 0-6.3%), and 11.3% (95% CI, 0.1-2.1.2%), respectively. There were no type I or III endoleaks. CONCLUSIONS: The AFX2 endograft plays a safe and effective role in treatment of infrarenal aortic pathologies that may be otherwise more technically challenging for traditional modular, bifurcated devices.

Exposure to Trichloroethylene Metabolite S-(1,2-Dichlorovinyl)-L-cysteine Causes Compensatory Changes to Macronutrient Utilization and Energy Metabolism in Placental HTR-8/SVneo Cells.

Trichloroethylene (TCE) is a widespread environmental contaminant following decades of use as an industrial solvent, improper disposal, and remediation challenges. Consequently, TCE exposure continues to constitute a risk to human health. Despite epidemiological evidence associating exposure with adverse birth outcomes, the effects of TCE and its metabolite S-(1, 2-dichlorovinyl)-L-cysteine (DCVC) on the placenta remain undetermined. Flexible and efficient macronutrient and energy metabolism pathway utilization is essential for placental cell physiological adaptability. Because DCVC is known to compromise cellular energy status and disrupt energy metabolism in renal proximal tubular cells, this study investigated the effects of DCVC on cellular energy status and energy metabolism pathways in placental cells. Human extravillous trophoblast cells, HTR-8/SVneo, were exposed to 5-20 µM DCVC for 6 or 12 h. After establishing concentration and exposure duration thresholds for DCVC-induced cytotoxicity, targeted metabolomics was used to evaluate overall energy status and metabolite concentrations from energy metabolism pathways. The data revealed glucose metabolism perturbations including a time-dependent accumulation of glucose-6-phosphate+frutose-6-phosphate (G6P+F6P) as well as independent shunting of glucose intermediates that diminished with time, with modest energy status decline but in the absence of significant changes in ATP concentrations. Furthermore, metabolic profiling suggested that DCVC stimulated compensatory utilization of glycerol, lipid, and amino acid metabolism to provide intermediate substrates entering downstream in the glycolytic pathway or the tricarboxylic acid cycle. Lastly, amino acid deprivation increased susceptibility to DCVC-induced cytotoxicity. Taken together, these results suggest that DCVC caused metabolic perturbations necessitating adaptations in macronutrient and energy metabolism pathway utilization to maintain adequate ATP levels.

Identification of environmental chemicals targeting miscarriage genes and pathways using the comparative toxicogenomics database.

BACKGROUND: Miscarriage is a prevalent public health issue and many events occur before women are aware of their pregnancy, complicating research design. Thus, risk factors for miscarriage are critically understudied. Our goal was to identify environmental chemicals with a high number of interactions with miscarriage genes, based on known toxicogenomic responses. METHODS: We used miscarriage (MeSH: D000022) and chemical gene lists from the Comparative Toxicogenomics Database in human, mouse, and rat. We assessed enrichment for gene ontology biological processes among the miscarriage genes. We prioritized chemicals (n = 25) found at Superfund sites or in the blood or urine pregnant women. For chemical-disease gene sets of sufficient size (n = 13 chemicals, n = 20 comparisons), chi-squared enrichment tests and proportional reporting ratios (PRR) were calculated. We cross-validated enrichment results. RESULTS: Miscarriage was annotated with 121 genes and overrepresented in inflammatory response (q = 0.001), collagen metabolic process (q = 1 × 10-13), cell death (q = 0.02), and vasculature development (q = 0.005) pathways. The number of unique genes annotated to a chemical ranged from 2 (bromacil) to 5607 (atrazine). In humans, all chemicals tested were highly enriched for miscarriage gene overlap (all p < 0.001; parathion PRR = 7, cadmium PRR = 6.5, lead PRR = 3.9, arsenic PRR = 3.5, atrazine PRR = 2.8). In mice, highest enrichment (p < 0.001) was observed for naphthalene (PRR = 16.1), cadmium (PRR = 12.8), arsenic (PRR = 11.6), and carbon tetrachloride (PRR = 7.7). In rats, we observed highest enrichment (p < 0.001) for cadmium (PRR = 8.7), carbon tetrachloride (PRR = 8.3), and dieldrin (PRR = 5.3). Our findings were robust to 1000 permutations each of variable gene set sizes. CONCLUSION: We observed chemical gene sets (parathion, cadmium, naphthalene, carbon tetrachloride, arsenic, lead, dieldrin, and atrazine) were highly enriched for miscarriage genes. Exposures to chemicals linked to miscarriage, and thus linked to decreased probability of live birth, may limit the inclusion of fetuses susceptible to adverse birth outcomes in epidemiology studies. Our findings have critical public health implications for successful pregnancies and the interpretation of adverse impacts of environmental chemical exposures on pregnancy.

Group B streptococcus activates transcriptomic pathways related to premature birth in human extraplacental membranes in vitro.

Streptococcus agalactiae (group B streptococcus [GBS]) infection in pregnant women is the leading cause of infectious neonatal morbidity and mortality in the United States. Although inflammation during infection has been associated with preterm birth, the contribution of GBS to preterm birth is less certain. Moreover, the early mechanisms by which GBS interacts with the gestational tissue to affect adverse pregnancy outcomes are poorly understood. We hypothesized that short-term GBS inoculation activates pathways related to inflammation and premature birth in human extraplacental membranes. We tested this hypothesis using GBS-inoculated human extraplacental membranes in vitro. In agreement with our hypothesis, a microarray-based transcriptomics analysis of gene expression changes in GBS-inoculated membranes revealed that GBS activated pathways related to inflammation and preterm birth with significant gene expression changes occurring as early as 4 h postinoculation. In addition, pathways related to DNA replication and repair were downregulated with GBS treatment. Conclusions based on our transcriptomics data were further supported by responses of prostaglandin E2 (PGE2), and matrix metalloproteinases 1 (MMP1) and 3 (MMP3), all of which are known to be involved in parturition and premature rupture of membranes. These results support our initial hypothesis and provide new information on molecular targets of GBS infection in human extraplacental membranes.

Trichloroethylene metabolite S-(1,2-dichlorovinyl)-l-cysteine induces lipid peroxidation-associated apoptosis via the intrinsic and extrinsic apoptosis pathways in a first-trimester placental cell line.

Trichloroethylene (TCE), a prevalent environmental contaminant, is a potent renal and hepatic toxicant through metabolites such as S-(1, 2-dichlorovinyl)-l-cysteine (DCVC). However, effects of TCE on other target organs such as the placenta have been minimally explored. Because elevated apoptosis and lipid peroxidation in placenta have been observed in pregnancy morbidities involving poor placentation, we evaluated the effects of DCVC exposure on apoptosis and lipid peroxidation in a human extravillous trophoblast cell line, HTR-8/SVneo. We exposed the cells in vitro to 10-100µM DCVC for various time points up to 24h. Following exposure, we measured apoptosis using flow cytometry, caspase activity using luminescence assays, gene expression using qRT-PCR, and lipid peroxidation using a malondialdehyde quantification assay. DCVC significantly increased apoptosis in time- and concentration-dependent manners (p<0.05). DCVC also significantly stimulated caspase 3, 7, 8 and 9 activities after 12h (p<0.05), suggesting that DCVC stimulates the activation of both the intrinsic and extrinsic apoptotic signaling pathways simultaneously. Pre-treatment with the tBID inhibitor Bl-6C9 partially reduced DCVC-stimulated caspase 3 and 7 activity, signifying crosstalk between the two pathways. Additionally, DCVC treatment increased lipid peroxidation in a concentration-dependent manner. Co-treatment with the antioxidant peroxyl radical scavenger (±)-α-tocopherol attenuated caspase 3 and 7 activity, suggesting that lipid peroxidation mediates DCVC-induced apoptosis in extravillous trophoblasts. Our findings suggest that DCVC-induced apoptosis and lipid peroxidation in extravillous trophoblasts could contribute to poor placentation if similar effects occur in vivo in response to TCE exposure, indicating that further studies into this mechanism are warranted.

Stoichiometry of the eIF2B complex is maintained by mutual stabilization of subunits.

The eukaryotic translation initiation factor eIF2B is a multi-subunit complex with a crucial role in the regulation of global protein synthesis in the cell. The complex comprises five subunits, termed α through ε in order of increasing size, arranged as a heterodecamer with two copies of each subunit. Regulation of the co-stoichiometric expression of the eIF2B subunits is crucial for the proper function and regulation of the eIF2B complex in cells. We have investigated the control of stoichiometric eIF2B complexes through mutual stabilization of eIF2B subunits. Our data show that the stable expression of the catalytic eIF2Bε subunit in human cells requires co-expression of eIF2Bγ. Similarly, stable expression of eIF2Bδ requires both eIF2Bß and eIF2Bγ+ε. The expression of these subunits decreases despite there being no change in either the levels or the translation of their mRNAs. Instead, these subunits are targeted for degradation by the ubiquitin-proteasome system. The data allow us to propose a model for the formation of stoichiometric eIF2B complexes which can ensure their stoichiometric incorporation into the holocomplex.

Placental cell conditioned media modifies hematopoietic stem cell transcriptome invitro.

INTRODUCTION: Hematopoietic stem cells are cells that differentiate into blood cell types. Although the placenta secretes hormones, proteins and other factors important for maternal/fetal health, cross-talk between placental and hematopoietic stem cells is poorly understood. Moreover, toxicant impacts on placental-hematopoietic stem cell communication is understudied. The goals of this study were to determine if factors secreted from placental cells alter transcriptomic responses in hematopoietic stem cells and if monoethylhexyl phthalate (MEHP), the bioactive metabolite of the pollutant diethylhexyl phthalate, modifies these effects. METHODS: We used K-562 and BeWo cells as in vitro models of hematopoietic stem cells and placental syncytiotrophoblasts, respectively. We treated K-562 cells with medium conditioned by incubation with BeWo cells, medium conditioned with BeWo cells treated with 10 µM MEHP for 24 h, or controls treated with unconditioned medium. We extracted K-562 cell RNA, performed RNA sequencing, then conducted differential gene expression and pathway analysis. RESULTS: Relative to controls, K-562 cells treated with BeWo cell conditioned medium differentially expressed 173 genes (FDR<0.05 and fold-change>2.0), including 2.4-fold upregulatation of tropomyosin 4 (TPM4, a cytoskeletal regulator involved in processes such as cell morphology and migration) and 3.3-fold upregulatation of sphingosine-1-phosphate receptor 3 (S1PR3, a mediator of myeloid cell differentiation and inflammatory responses). Upregulated genes were enriched for pathways including stem cell maintenance, cell proliferation and immune processes. Downregulated genes were enriched for terms involved in protein translation and transcriptional regulation. MEHP treatment differentially expressed eight genes (FDR<0.05), including genes involved in lipid metabolism (e.g., Perilipin 2, fold-change: 1.4; Carnitine Palmitoyltransferase 1A, fold-change: 1.4). DISCUSSION: K-562 cells, a model of hematopoietic stem cells, are responsive to media conditioned by placental cells, potentially impacting pathways like stem cell maintenance.

The antioxidant N-acetyl cysteine inhibits cytokine and prostaglandin release in human fetal membranes stimulated ex vivo with lipoteichoic acid or live group B streptococcus.

BACKGROUNDS: Infection during pregnancy is a significant public health concern due to the increased risk of adverse birth outcomes. Group B Streptococcus or Streptococcus agalactiae (GBS) stands out as a major bacterial cause of neonatal morbidity and mortality. We aimed to explore the involvement of reactive oxygen species (ROS) and oxidative stress pathways in pro-inflammatory responses within human fetal membrane tissue, the target tissue of acute bacterial chorioamnionitis. METHODS: We reanalyzed transcriptomic data from fetal membrane explants inoculated with GBS to assess the impact of GBS on oxidative stress and ROS genes/pathways. We conducted pathway enrichment analysis of transcriptomic data using the Database for Annotation, Visualization and Integrated Discovery (DAVID), a web-based functional annotation/pathway enrichment tool. Subsequently, we conducted ex vivo experiments to test the hypothesis that antioxidant treatment could inhibit pathogen-stimulated inflammatory responses in fetal membranes. RESULTS: Using DAVID analysis, we found significant enrichment of pathways related to oxidative stress or ROS in GBS-inoculated human fetal membranes, for example, "Response to Oxidative Stress" (FDR = 0.02) and "Positive Regulation of Reactive Oxygen Species Metabolic Process" (FDR = 2.6*10-4 ). There were 31 significantly changed genes associated with these pathways, most of which were upregulated after GBS inoculation. In ex vivo experiments with choriodecidual membrane explants, our study showed that co-treatment with N-acetylcysteine (NAC) effectively suppressed the release of pro-inflammatory cytokines (IL-6, IL-8, TNF-α) and prostaglandin PGE2, compared to GBS-treated explants (p < .05 compared to GBS-treated samples without NAC co-treatment). Furthermore, NAC treatment inhibited the release of cytokines and PGE2 stimulated by lipoteichoic acid (LTA) and lipopolysaccharide (LPS) in whole membrane explants (p < .05 compared to LTA or LPS-treated samples without NAC co-treatment). CONCLUSIONS: Our study sheds light on the potential roles of ROS in governing the innate immune response to GBS infection, offering insights for developing strategies to mitigate GBS-related adverse outcomes.

Pharmaceutical modulation of canonical Wnt signaling in multipotent stromal cells for improved osteoinductive therapy.

Human mesenchymal stem cells (hMSCs) from bone marrow are regarded as putative osteoblast progenitors in vivo and differentiate into osteoblasts in vitro. Positive signaling by the canonical wingless (Wnt) pathway is critical for the differentiation of MSCs into osteoblasts. In contrast, activation of the peroxisome proliferator-activated receptor-gamma (PPARgamma)-mediated pathway results in adipogenesis. We therefore compared the effect of glycogen-synthetase-kinase-3beta (GSK3beta) inhibitors and PPARgamma inhibitors on osteogenesis by hMSCs. Both compounds altered the intracellular distribution of beta-catenin and GSK3beta in a manner consistent with activation of Wnt signaling. With osteogenic supplements, the GSK3beta inhibitor 6-bromo-indirubin-3'-oxime (BIO) and the PPARgamma inhibitor GW9662 (GW) enhanced early osteogenic markers, alkaline phosphatase (ALP), and osteoprotegerin (OPG) by hMSCs and transcriptome analysis demonstrated up-regulation of genes encoding bone-related structural proteins. At higher doses of the inhibitors, ALP levels were attenuated, but dexamethasone-induced biomineralization was accelerated. When hMSCs were pretreated with BIO or GW and implanted into experimentally induced nonself healing calvarial defects, GW treatment substantially increased the capacity of the cells to repair the bone lesion, whereas BIO treatment had no significant effect. Further investigation indicated that unlike GW, BIO induced cell cycle inhibition in vitro. Furthermore, we found that GW treatment significantly reduced expression of chemokines that may exacerbate neutrophil- and macrophage-mediated cell rejection. These data suggest that use of PPARgamma inhibitors during the preparation of hMSCs may enhance the capacity of the cells for osteogenic cytotherapy, whereas adenine analogs such as BIO can adversely affect the viability of hMSC preparations in vitro and in vivo.

American Indians travel great distances for obstetrical care: Examining rural and racial disparities.

Rural, American Indian/Alaska Native (AI/AN) people, a population at elevated risk for complex pregnancies, have limited access to risk-appropriate obstetric care. Obstetrical bypassing, seeking care at a non-local obstetric unit, is an important feature of perinatal regionalization that can alleviate some challenges faced by this rural population, at the cost of increased travel to give birth. Data from five years (2014-2018) of birth certificates from Montana, along with the 2018 annual survey of the American Hospital Association (AHA) were used in logistic regression models to identify predictors of bypassing, with ordinary least squares regression models used to predict factors associated with the distance (in miles) birthing people drove beyond their local obstetric unit to give birth. Logit analyses focused on hospital-based births to Montana residents delivered during this time period (n = 54,146 births). Distance analyses focused on births to individuals who bypassed their local obstetric unit to deliver (n = 5,991 births). Individual-level predictors included maternal sociodemographic characteristics, location, perinatal health characteristics, and health care utilization. Facility-related measures included level of obstetric care of the closest and delivery hospitals, and distance to the closest hospital-based obstetric unit. Findings suggest that birthing people living in rural areas and on American Indian reservations were more likely to bypass to give birth, with bypassing likelihood depending on health risk, insurance, and rurality. AI/AN and reservation-dwelling birthing people traveled significantly farther when bypassing. Findings highlight that distance traveled was even farther for AI/AN people facing pregnancy health risks (23.8 miles farther than White people with pregnancy risks) or when delivering at facilities offering complex care (14-44 miles farther than White people). While bypassing may connect rural birthing people to more risk-appropriate care, rural and racial inequities in access persist, with rural, reservation-dwelling AI/AN birthing people experiencing greater likelihood of bypassing and traveling greater distances when bypassing.

Placental single cell transcriptomics: Opportunities for endocrine disrupting chemical toxicology.

The placenta performs essential biologic functions for fetal development throughout pregnancy. Placental dysfunction is at the root of multiple adverse birth outcomes such as intrauterine growth restriction, preeclampsia, and preterm birth. Exposure to endocrine disrupting chemicals during pregnancy can cause placental dysfunction, and many prior human studies have examined molecular changes in bulk placental tissues. Placenta-specific cell types, including cytotrophoblasts, syncytiotrophoblasts, extravillous trophoblasts, and placental resident macrophage Hofbauer cells play unique roles in placental development, structure, and function. Toxicant-induced changes in relative abundance and/or impairment of these cell types likely contribute to placental pathogenesis. Although gene expression insights gained from bulk placental tissue RNA-sequencing data are useful, their interpretation is limited because bulk analysis can mask the effects of a chemical on individual populations of placental cells. Cutting-edge single cell RNA-sequencing technologies are enabling the investigation of placental cell-type specific responses to endocrine disrupting chemicals. Moreover, in situ bioinformatic cell deconvolution enables the estimation of cell type proportions in bulk placental tissue gene expression data. These emerging technologies have tremendous potential to provide novel mechanistic insights in a complex heterogeneous tissue with implications for toxicant contributions to adverse pregnancy outcomes.

Placental Cell Conditioned Media Modifies Hematopoietic Stem Cell Transcriptome In Vitro.

Background: Hematopoietic stem cells are cells that differentiate into all blood cell types. Although the placenta secretes hormones, proteins and other factors important for maternal and fetal health, cross-talk between placental cells and hematopoietic stem cells is poorly understood. Moreover, toxicant impacts on placental-hematopoietic stem cell communication is understudied. The goals of this study were to determine if factors secreted from placental cells alter transcriptomic responses in hematopoietic stem cells and if monoethylhexyl phthalate (MEHP), the bioactive metabolite of the pollutant diethylhexyl phthalate, modifies these effects. Methods: We used K-562 and BeWo cells as in vitro models of hematopoietic stem cells and placental syncytiotrophoblasts, respectively. We treated K-562 cells with medium conditioned by incubation with BeWo cells, medium conditioned with BeWo cells treated with 10 µM MEHP for 24 hours, or controls treated with unconditioned medium. We extracted K-562 cell RNA, performed RNA sequencing, then conducted differential gene expression and pathway analysis by treatment group. Results: Relative to controls, K-562 cells treated with BeWo cell conditioned medium differentially expressed 173 genes (FDR<0.05 and fold-change>2.0), including 2.4 fold upregulatation of TPM4 and 3.3 fold upregulatation of S1PR3. Upregulated genes were enriched for pathways including stem cell maintenance, cell proliferation and immune processes. Downregulated genes were enriched for terms involved in protein translation and transcriptional regulation. MEHP treatment differentially expressed eight genes (FDR<0.05), including genes involved in lipid metabolism (PLIN2, fold-change: 1.4; CPT1A, fold-change: 1.4). Conclusion: K-562 cells, a model of hematopoietic stem cells, are responsive to media conditioned by placental cells, potentially impacting pathways like stem cell maintenance and proliferation.

A dynamic in vitro developing testis model reflects structures and functions of testicular development in vivo.

To better define appropriate applications of our 3-dimensional testicular co-culture as a model for reproductive toxicology, we evaluated the ability of the model to capture structural and functional elements that can be targeted by reproductive toxicants. Testicular co-cultures were prepared from postnatal day 5 male rats and cultured with a Matrigel overlay. Following a 2-day acclimation period, we characterized functional pathway dynamics by evaluating morphology, protein expression, testosterone concentrations, and global gene expression at a range of timepoints from experimental days 0-21. Western blotting confirmed expression of Sertoli cell, Leydig cell, and spermatogonial cell-specific protein markers. Testosterone detected in cell culture media indicates active testosterone production. Quantitative pathway analysis identified Gene Ontology biological processes enriched among genes significantly changing over the course of 21 days. Processes enriched among genes significantly increasing through time include general developmental processes (morphogenesis, tissue remodeling, etc.), steroid regulation, Sertoli cell development, immune response, and stress and apoptosis. Processes enriched among genes significantly decreasing over time include several related to male reproductive development (seminiferous tubule development, male gonad development, Leydig cell differentiation, Sertoli cell differentiation), all of which appear to peak in expression between days 1 and 5 before decreasing at later timepoints. This analysis provides a temporal roadmap for specific biological process of interest for reproductive toxicology in the model and anchors the model to sensitive phases of in vivo development, helping to define the relevance of the model for in vivo processes.

Prevalence and Characteristics of Patients with Median Arcuate Ligament Syndrome in a Cohort Diagnosed with Celiac Artery Compression.

BACKGROUND: Median arcuate ligament syndrome (MALS) is a frequent differential diagnosis in patients with postprandial abdominal symptoms, but diagnosis remains challenging. The aim of this study was to identify characteristics of patients who had MALS compared with non-MALS patients among a cohort of patients diagnosed with celiac artery compression (CAC). STUDY DESIGN: An IRB-approved retrospective chart review (2000 to 2021) of patients at our institution with a discharge diagnosis of CAC was performed. Medical record review for clinical symptoms and findings consistent with MALS was performed. RESULTS: Two hundred ninety-three patients with a diagnosis of CAC were identified; 59.7% were women, and average age was 63.9 ± 20.2 years. Sixty-nine (23.5%) patients with CAC had MALS. There were no significant differences in sex or race between MALS and non-MALS patients, but MALS patients were younger (55.7 vs 68.1, p < 0.001). There was no significant difference in gastrointestinal comorbidities between the 2 groups. Patients with MALS were less likely to have diabetes (12.5% vs 26.9%), renal disease (4.6% vs 8.2%), hypertension (41.5% vs 70.3%), mesenteric atherosclerotic disease (14% vs 61.9%), and peripheral artery disease (15.0% vs 39.7%). CONCLUSIONS: We demonstrate a novel observation that MALS patients tend to have fewer atherosclerotic characteristics than non-MALS patients with CAC. Patients in our study with MALS were more likely to be younger, women, and presenting with epigastric pain. MALS patients had a significantly lower incidence of diabetes, hypertension, renal disease, mesenteric artery disease, and peripheral arterial disease compared with the non-MALS group. An important clinically relevant feature of MALS patients may be their lack of atherosclerotic phenotype compared with non- MALS patients with CAC.

Sexually concordant and dimorphic transcriptional responses to maternal trichloroethylene and/or N-acetyl cysteine exposure in Wistar rat placental tissue.

Numerous Superfund sites are contaminated with the volatile organic chemical trichloroethylene (TCE). In women, exposure to TCE in pregnancy is associated with reduced birth weight. Our previous study reported that TCE exposure in pregnant rats decreased fetal weight and elevated oxidative stress biomarkers in placentae, suggesting placental injury as a potential mechanism of TCE-induced adverse birth outcomes. In this study, we investigated if co-exposure with the antioxidant N-acetylcysteine (NAC) attenuates TCE exposure effects on RNA expression. Timed-pregnant Wistar rats were exposed orally to 480 mg TCE/kg/day on gestation days 6-16. Exposure of 200 mg NAC/kg/day alone or as a pre/co-exposure with TCE occurred on gestation days 5-16 to stimulate antioxidant genes prior to TCE exposure. Tissue was collected on gestation day 16. In male and female placentae, we evaluated TCE- and/or NAC-induced changes to gene expression and pathway enrichment analyses using false discovery rate (FDR) and fold-change criteria. In female placentae, exposure to TCE caused significant differential expression 129 genes while the TCE+NAC altered 125 genes, compared with controls (FDR< 0.05 + fold-change >1). In contrast, in male placentae TCE exposure differentially expressed 9 genes and TCE+NAC differentially expressed 35 genes, compared with controls (FDR< 0.05 + fold-change >1). NAC alone did not significantly alter gene expression in either sex. Differentially expressed genes observed with TCE exposure were enriched in mitochondrial biogenesis and oxidative phosphorylation pathways in females whereas immune system pathways and endoplasmic reticulum stress pathways were differentially expressed in both sexes (FDR<0.05). TCE treatment was differentially enriched for genes regulated by the transcription factors ATF6 (both sexes) and ATF4 (males only), indicating a cellular condition triggered by misfolded proteins during endoplasmic reticulum stress. This study demonstrates novel genes and pathways involved in TCE-induced placental injury and showed antioxidant co-treatment largely did not attenuate TCE exposure effects.

Core musculature co-contraction during suspension training exercises.

Suspension training reportedly enhances core musculature co-contraction. This study investigated whether the use of a suspension trainer increases core musculature co-activation during exercises vs. its floor counterpart. Participants were 25 healthy volunteers (16 men, 9 women; age: 27.24 ± 4.02 years). Wireless electromyography electrodes were placed bilaterally at the rectus abdominis (RA), erector spinae (ES), and abdominal obliques (OB). Test order (push-up, bridge, and prone plank) was randomized (exercise and condition) with a 3-min rest period between tests. Co-contraction ratios between muscle groups were estimated by root mean square. Ratios (RA/ES, RA/OB, ES/OB) were analyzed using paired t-tests (P ≤ .05). For all floor exercises, co-contraction of core musculature was significantly higher than suspension trainer. During suspension training, perturbations due to increased agonist activation without similar increases in antagonists may be too intense for untrained or injured individuals. Individuals lacking muscle control to recruit muscles concurrently may benefit from mastering traditional floor exercises to promote joint stiffness and stability before suspension trainer exercises.