ROLES OF SULFOTRANSFERASE ENZYMES IN TRANS-PLACENTAL DISPOSITION

The trophoblast cell layer constitutes the rate-determining barrier for trans-placental transfer. Several isoforms of the sulfotransferase enzymes are functional in placenta but there is only limited information available on the utility of cultured trophoblast cells for studying placental sulfation. We examined the expression and activities of four sulfotransferase isoforms (SULT1A1, SULT1A3, SULT1E1, and SULT2A1) in primary cytotrophoblast cells and the trophoblast-like BeWo cell line. These isoforms have been reported to be functional in human placenta. Our results indicated that the phenolic sulfotransferase isoforms, SULT1A1 and SULT1A3, are functional in BeWo, as well as in the primary cytotrophoblast cells. SULT2A1 and SULT1E1 are not functional in either cell type. We also found that chronic exposure to the industrial chemical bisphenol A inhibited SULT1A1 activity. A U-shaped dose-response curve was observed with inhibition (~30-40%) being observed only at intermediate concentrations (10-100 nM). These results suggested that trophoblast cells may be used as a suitable in vitro tool to determine the effect of endogenous or exogenous substances on placental sulfotransferase activity. Altered metabolic activity has the chance of altering fetal exposure to drugs and other substances in the maternal circulation. Studies performed in BeWo cells also suggested that one of the roles of the placental efflux transporters is the elimination of sulfate metabolites. The multidrug resistance-associated proteins (MRPs) played a major role in the elimination of 4-nitrophenyl sulfate and acetaminophen sulfate across the basolateral (fetal-facing) and apical (maternal-facing) trophoblast membranes respectively. The breast cancer resistance protein (BCRP) played a minor role in the elimination of these two sulfate conjugates across the apical membrane

Full-scale Deeply Supervised Attention Network for Segmenting COVID-19 Lesions

Automated delineation of COVID-19 lesions from lung CT scans aids the diagnosis and prognosis for patients. The asymmetric shapes and positioning of the infected regions make the task extremely difficult. Capturing information at multiple scales will assist in deciphering features, at global and local levels, to encompass lesions of variable size and texture. We introduce the Full-scale Deeply Supervised Attention Network (FuDSA-Net), for efficient segmentation of corona-infected lung areas in CT images. The model considers activation responses from all levels of the encoding path, encompassing multi-scalar features acquired at different levels of the network. This helps segment target regions (lesions) of varying shape, size and contrast. Incorporation of the entire gamut of multi-scalar characteristics into the novel attention mechanism helps prioritize the selection of activation responses and locations containing useful information. Determining robust and discriminatory features along the decoder path is facilitated with deep supervision. Connections in the decoder arm are remodeled to handle the issue of vanishing gradient. As observed from the experimental results, FuDSA-Net surpasses other state-of-the-art architectures; especially, when it comes to characterizing complicated geometries of the lesions

Expression and Functional Activities of Selected Sulfotransferase Isoforms in BeWo Cells and Primary Cytotrophoblast Cells

Several cytosolic sulfotransferase enzyme isoforms are functional in placenta but there is limited information available on the utility of cultured trophoblast cells for studying sulfation. The trophoblast cell layer constitutes the rate-determining barrier for trans-placental transfer. The objective of this work was to examine the mRNA expression and enzyme activities of four sulfotransferase isoforms reported to be functional in human placenta (SULT1A1, SULT1A3, SULT1E1, and SULT2A1) in primary cytotrophoblast cells and the trophoblast-like BeWo cell line. Reverse transcription polymerase chain reaction (RT-PCR) was performed to determine mRNA expression. Enzyme activities were assessed using the following substrates: 4-nitrophenol for SULT1A1, dopamine for SULT1A3, 17β-estradiol for SULT1E1, and dehydroepiandrosterone for SULT2A1. For 4-nitrophenol and dopamine sulfation, apparent Km values, response to inhibitors (2,6-dichloro-4-nitrophenol and sodium chloride), and thermal stability profiles indicated that 4-nitrophenol and dopamine sulfation in BeWo cells were being mediated by SULT1A1 and SULT1A3, respectively. SULT1A1 and SULT1A3 were also functional in the cytotrophoblast cells. Both at the protein and at the mRNA levels, SULT1A1 was more abundant in BeWo cells in comparison to the primary cytotrophoblast cells. SULT1E1 and SULT2A1 mRNA were not detected in the cytotrophoblasts. SULT1E1 mRNA was weakly expressed in BeWo but there was negligible functional activity. Although SULT2A1 mRNA was abundantly expressed in BeWo, Western blot and enzyme activities revealed that the protein is not expressed in BeWo cells. The results suggest that the BeWo cells and the cytotrophoblast cells can be used to examine the roles of SULT1A1 and SULT1A3 in placental metabolism

Multidrug resistance-associated protein (MRP) isoforms and the breast cancer resistance protein (BCRP) mediate sulfate conjugate efflux out of BeWo cells

The breast cancer resistance protein (BCRP) and the multidrug resistance-associated proteins (MRPs) have the ability to eliminate sulfate conjugates but it is not known if this constitutes one of their roles in the placenta. To determine this, the BeWo cell line was used as a model of placental trophoblast cells and we examined the fate of two common sulfotransferase substrates, 4-nitrophenol and acetaminophen. At 0.5–200 μM, acetaminophen sulfate did not alter the accumulation of the BCRP substrates BODIPY FL prazosin or mitoxantrone in BeWo monolayers indicating a lack of interaction of BCRP with acetaminophen sulfate. 4-nitrophenyl sulfate increased the accumulation of BODIPY FL prazosin only at 200 μM, indicating it to be a BCRP inhibitor at high concentrations. Efflux studies and bidirectional transport studies examining the effect of BCRP/MRP inhibitors on the efflux of intracellularly generated 4-nitrophenyl sulfate and acetaminophen sulfate, indicated that one or more of the MRP isoforms played a major role in the elimination of 4-nitrophenyl sulfate and acetaminophen sulfate across the basolateral (fetal-facing) and apical (maternal-facing) trophoblast membranes respectively. BCRP played only a minor role in the elimination of these two sulfate conjugates across the apical membrane. Our study shows that a yet undetermined role of trophoblast efflux transporters is the elimination of sulfate conjugate

MRP isoforms and BCRP mediate sulfate conjugate efflux out of BeWo cells

The breast cancer resistance protein (BCRP) and the multidrug resistance-associated proteins (MRPs) have the ability to eliminate sulfate conjugates but it is not known if this constitutes one of their roles in the placenta. To determine this, the BeWo cell line was used as a model of placental trophoblast cells and we examined the mechanisms of elimination of two common sulfotransferase substrates, 4-nitrophenol and acetaminophen. At 0.5–200 μM, neither 4-nitrophenyl sulfate nor acetaminophen sulfate affected the accumulation of the BCRP substrates BODIPY FL prazosin or mitoxantrone in BeWo monolayers, indicating a lack of interaction of BCRP with the sulfates. Efflux studies and bidirectional transport studies examining the effect of BCRP/MRP inhibitors on the efflux of intracellularly generated 4-nitrophenyl sulfate and acetaminophen sulfate, indicated that one or more of the MRP isoforms play a major role in the elimination of 4-nitrophenyl sulfate and acetaminophen sulfate across the basolateral (fetal-facing) and apical (maternal-facing) membranes respectively. BCRP played a minor role in the elimination of these two sulfate conjugates across the apical membrane. Our study shows that a yet undetermined role of trophoblast efflux transporters is the elimination of sulfate conjugates

QSPR Modeling of Odor Threshold of Aliphatic Alcohols Using Extended Topochemical Atom (ETA) Indices

The present work establishes a quantitative structure-property relationship (QSPR) between top¬ochemical features and odor threshold (OT) of aliphatic alcohols. A data set of 53 aliphatic alcohols was chosen for the analysis employing different chemometric techniques, among which, genetic function ap¬proximation with spline option (GFA-spline) showed the most acceptable results in terms of internal and external validation metric values. The extended topochemical atom (ETA) indices, developed by the pre¬sent authors’ group, were considered as descriptors for model development. Additionally, selected non-ETA descriptors were also tried for model development. It was observed that the models with ETA indi¬ces significantly surpass the predictive ability of the models developed using other descriptors. The final model suggests that molecular branching and electronic parameters significantly influence the odor poten-cy of the molecules. Additionally, increased lipophilicity and reduced electronegativity increase the odor-ant property. The model thus developed may effectively be used for prediction of odor threshold of any untested aliphatic alcohols. (doi: 10.5562/cca2284

DNA replication during intra-erythrocytic stages of human malarial parasite Plasmodium falciparum

Plasmodium falciparum, the causative agent of the most virulent form of human malaria, has deservingly held the candidature of being one of the most studied human pathogens. Here we attempt an overview of the studies probing one of the important aspects of DNA metabolism, that is, DNA replication in this parasite, focusing on the relatively well-characterized core components of chromosomal DNA replication in the asexual intra-erythrocytic stage

Functional dissection of the catalytic carboxyl-terminal domain of Origin Recognition Complex Subunit 1 (PfORC1) of the human malaria parasite Plasmodium falciparum

Origin Recognition Complex subunit 1 (ORC1) is essential for DNA replication in eukaryotes. The deadly human malaria parasite Plasmodium falciparum contains an ORC1/CDC6 homolog with several interesting domains at the catalytic carboxyl-terminal region that include a putative nucleoside triphosphate-binding and hydrolysis domain, a putative PCNA-Interacting-Protein (PIP) motif and an extreme C-terminal region that shows poor homology with other ORC1 homologs. Due to the unavailability of a dependable inducible gene expression system, it is difficult to study the structure and function of essential genes in Plasmodium. Using a genetic yeast complementation system and biochemical experiments, here we show that the putative PIP domain in ORC1 that facilitates in vitro physical interaction with PCNA is functional in both yeast (Saccharomyces cerevisiae) and Plasmodium in vivo, confirming its essential biological role in eukaryotes. Furthermore, despite having less sequence homology, the extreme C-terminal region can be swapped between S. cerevisiae and P. falciparum and it binds to DNA directly, suggesting a conserved role of this region in DNA replication. These results not only provide us a useful system to study the function of the essential genes in Plasmodium, they help us to identify the previously undiscovered unique features of replication proteins in general

The role of N-terminus of Plasmodium falciparum ORC1 in telomeric localization and var gene silencing

Plasmodium falciparum origin recognition complex 1 (ORC1) protein has been implicated in DNA replication and silencing var gene family. However, the mechanism and the domain structure of ORC1 related to the regulation of var gene family are unknown. Here we show that the unique N-terminus of PfORC1 (PfORC1N1–238) is targeted to the nuclear periphery in vivo and this region binds to the telomeric DNA in vitro due to the presence of a leucine heptad repeats. Like PfORC1N1–238, endogenous full length ORC1, was found to be associated with sub telomeric repeat regions and promoters of various var genes. Additionally, binding and propagation of ORC1 to telomeric and subtelomeric regions was severely compromised in PfSir2 deficient parasites suggesting the dependence of endogenous ORC1 on Sir2 for var gene regulation. This feature is not previously described for Plasmodium ORC1 and contrary to yeast Saccharomyces cerevisiae where ORC function as a landing pad for Sir proteins. Interestingly, the overexpression of ORC1N1–238 compromises the binding of Sir2 at the subtelomeric loci and var gene promoters consistent with de-repression of some var genes. These results establish role of the N-terminus of PfORC1 in heterochromatin formation and regulation of var gene expression in co-ordination with Sir2 in P. falciparum