Basement membrane ultrastructure and component localization data from uterine tissues during early mouse pregnancy

Basement membranes (BMs) are specialized extracellular scaffolds that provide architecture and modulate cell behaviors in tissues, such as fat, muscle, endothelium, endometrium, and decidua. Properties of BMs are maintained in homeostasis for most adult tissues. However, BM ultrastructure, composition, and localization are rapidly altered in select uterine tissues that are reprogrammed during pregnancy to enable early maternal-embryo interactions. Here, our data exhibit both static and dynamic BMs that were tracked in mouse uterine tissues during pre-, peri-, and postimplantation periods of pregnancy. The data exhibit spatial-temporal patterns of BM property regulation that coincide with the progression of adapted physiology. Further interpretation and discussion of these data in this article are described in the associated research article titled, “Embryo implantation triggers dynamic spatiotemporal expression of the basement membrane toolkit during uterine reprogramming” (C.R. Jones-Paris, S. Paria, T. Berg, J. Saus, G. Bhave, B.C. Paria, B.G. Hudson, 2016) [1]

High-Throughput Screening of Myometrial Calcium-Mobilization to Identify Modulators of Uterine Contractility.

The uterine myometrium (UT-myo) is a therapeutic target for preterm labor, labor induction, and postpartum hemorrhage. Stimulation of intracellular Ca2+-release in UT-myo cells by oxytocin is a final pathway controlling myometrial contractions. The goal of this study was to develop a dual-addition assay for high-throughput screening of small molecular compounds, which could regulate Ca2+-mobilization in UT-myo cells, and hence, myometrial contractions. Primary murine UT-myo cells in 384-well plates were loaded with a Ca2+-sensitive fluorescent probe, and then screened for inducers of Ca2+-mobilization and inhibitors of oxytocin-induced Ca2+-mobilization. The assay exhibited robust screening statistics (Z´ = 0.73), DMSO-tolerance, and was validated for high-throughput screening against 2,727 small molecules from the Spectrum, NIH Clinical I and II collections of well-annotated compounds. The screen revealed a hit-rate of 1.80% for agonist and 1.39% for antagonist compounds. Concentration-dependent responses of hit-compounds demonstrated an EC50 less than 10μM for 21 hit-antagonist compounds, compared to only 7 hit-agonist compounds. Subsequent studies focused on hit-antagonist compounds. Based on the percent inhibition and functional annotation analyses, we selected 4 confirmed hit-antagonist compounds (benzbromarone, dipyridamole, fenoterol hydrobromide and nisoldipine) for further analysis. Using an ex vivo isometric contractility assay, each compound significantly inhibited uterine contractility, at different potencies (IC50). Overall, these results demonstrate for the first time that high-throughput small-molecules screening of myometrial Ca2+-mobilization is an ideal primary approach for discovering modulators of uterine contractility

Co-occurrence of arsenic and fluoride in groundwater of semi-arid regions in Latin America: genesis, mobility and remediation

Several million people around the world are currently exposed to excessive amounts of arsenic (As) and fluoride (F) in their drinking water. Although the individual toxic effects of As and F have been analyzed, there are few studies addressing their co-occurrences and water treatment options. Several studies conducted in arid and semi-arid regions of Latin America show that the co-occurrences of As and F in drinking water are linked to the volcaniclastic particles in the loess or alluvium, alkaline pH, and limited recharge. The As and F contamination results from water–rock interactions and may be accelerated by geothermal and mining activities, as well as by aquifer over-exploitation. These types of contamination are particularly pronounced in arid and semi-arid regions, where high As concentrations often show a direct relationship with high F concentrations. Enrichment of F is generally related to fluorite dissolution and it is also associated with high Cl, Br, and V concentrations. The methods of As and F removal, such as chemical precipitation followed by filtration and reverse osmosis, are currently being used at different scales and scenarios in Latin America. Although such technologies are available in Latin America, it is still urgent to develop technologies and methods capable of monitoring and removing both of these contaminants simultaneously from drinking water, with a particular focus towards small-scale rural operations

Target proteins identified by multiple confirmed compounds.

<p>Target proteins identified by multiple confirmed compounds.</p

Hit-agonist identification during pilot screen.

<p>A, <i>left panel</i>. Representative image of realtime monitoring of Ca²⁺-mobilization from UT-myo cells following the compound addition (white arrow, <i>middle panel</i>). The two end columns of each plate were used for checkerboard analyses to determine the Z´-factor for each HTS assay, and received either 0.1% DMSO vehicle or 10uM atosiban during the compound addition. The inner 320 wells received 10μM of test compounds. An example of a “Hit”-agonist is highlighted (orange box). A, <i>right panel</i>. Examples of relative fluorescent recordings from autofluorescent test compounds. B. Plate heatmap of Ca²⁺-mobilization at the time following compound addition (dashed line, middle panel A). A representative hit-agonist compound (green well) is highlighted by orange box, while autofluorescent compounds are visualized as bright red wells. C. The average cutoff threshold for hit-agonists was 5.85±1.59% stimulation, based on 3*MAD from median (refer to “Materials and Methods” section).</p

Assessment of primary murine UT-myo cell homogeneity.

<p>A. Representative photomicrograph of UT-myo cells prior to dissociation and use in Ca²⁺-mobilization or immunofluorescent staining. Representative photomicrograph of UT-myo cells (B) and uterine myometrium (C) stained with smooth muscle cell markers, alpha-SMA (red) and calponin (green), and DAPI (blue). UT-myo cells and whole-mount uterine tissue were collected from day 19 of mouse pregnancy. The placenta and embryo were removed from whole-mount tissue sections.</p

Capacity of compounds to inhibit <i>ex vivo</i> uterine myometrial contractility.

<p>Capacity of compounds to inhibit <i>ex vivo</i> uterine myometrial contractility.</p

Concentration-response effect of confirmed hit-compounds.

<p>A. Representative compound titrations performed to examine the potency of confirmed hit-agonists and hit-antagonists on UT-myo native and OT-induced Ca²⁺-mobilization. B. Additionally, compound titrations for hit-compounds were performed using commercially purchased compounds. A 10-point three-fold titration of confirmed hit-compounds were added during the compound addition of the Ca²⁺-mobilization assay. Data is shown as either mean±SEM %stimulation of Ca²⁺-mobilization or %inhibition of OT-induced Ca²⁺-mobilization. Non-linear regression was used to fit the data.</p

Compounds selected for <i>ex vivo</i> uterine myometrial organ bath contractility studies.

<p>¹ Values obtained from primary screen and retesting at 10μM concentration</p><p>Compounds selected for <i>ex vivo</i> uterine myometrial organ bath contractility studies.</p