Integrated microRNA and mRNA network analysis of the human myometrial transcriptome in the transition from quiescence to labor.

We conducted integrated transcriptomics network analyses of miRNA and mRNA interactions in human myometrium to identify novel molecular candidates potentially involved in human parturition. Myometrial biopsies were collected from women undergoing primary Cesarean deliveries in well-characterized clinical scenarios: (1) spontaneous term labor (TL, n = 5); (2) term nonlabor (TNL, n = 5); (3) spontaneous preterm birth (PTB) with histologic chorioamnionitis (PTB-HCA, n = 5); and (4) indicated PTB nonlabor (PTB-NL, n = 5). RNAs were profiled using RNA sequencing, and miRNA-target interaction networks were mined for key discriminatory subnetworks. Forty miRNAs differed between TL and TNL myometrium, while seven miRNAs differed between PTB-HCA vs. PTB-NL specimens; six of these were cross-validated using quantitative PCR. Based on the combined sequencing data, unsupervised clustering revealed two nonoverlapping cohorts that differed primarily by absence or presence of uterine quiescence, rather than gestational age or original clinical cohort. The intersection of differentially expressed miRNAs and their targets predicted 22 subnetworks with enriched representation of miR-146b-5p, miR-223-3p, and miR-150-5p among miRNAs, and of myocyte enhancer factor-2C (MEF2C) among mRNAs. Of four known MEF2 transcription factors, decreased MEF2A and MEF2C expression in women with uterine nonquiescence was observed in the sequencing data, and validated in a second cohort by quantitative PCR. Immunohistochemistry localized MEF2A and MEF2C to myometrial smooth muscle cells and confirmed decreased abundance with labor. Collectively, these results suggest altered MEF2 expression may represent a previously unrecognized process through which miRNAs contribute to the phenotypic switch from quiescence to labor in human myometrium.

Conformation-dependent anti-Aß monoclonal antibody signatures of disease status and severity in urine of women with preeclampsia.

Prior research has shown that urine of women with preeclampsia (PE) contains amyloid-like aggregates that are congophilic (exhibit affinity for the amyloidophilic dye Congo red) and immunoreactive with A11, a polyclonal serum against prefibrillar ß-amyloid oligomers, thereby supporting pathogenic similarity between PE and protein conformational disorders such as Alzheimer's and prion disease. The objective of this study was to interrogate PE urine using monoclonal antibodies with previously characterized A11-like epitopes. Over 100 conformation-dependent monoclonals were screened and three (mA11-09, mA11-89, and mA11-205) selected for further confirmation in 196 urine samples grouped as follows: severe features PE (sPE, n = 114), PE without severe features (mPE, n = 30), chronic hypertension (crHTN, n = 14) and normotensive pregnant control (P-CRL, n = 38). We showed that the selected conformation-specific monoclonals distinguished among patients with varying severities of PE from P-CRL and patients with crHTN. By use of latent class analysis (LCA) we identified three classes of subjects: Class 1 (n = 94) comprised patients whose urine was both congophilic and reactive with the monoclonals. These women were more likely diagnosed with early-onset sPE and had severe hypertension and proteinuria; Class 2 patients (n = 55) were negative for congophilia and against the antibodies. These were predominantly P-CRL and crHTN patients. Lastly, Class 3 patients (n = 48) were positive for urine congophilia, albeit at lower intensity, but negative for monoclonal immunoreactivities. These women were diagnosed primarily as mPE or late-onset sPE. Collectively, our study validates conformation-dependent Aß imunoreactivity of PE urine which in conjunction to urine congophilia may represent an additional indicator of disease severity.

AKR1B10 induces cell resistance to daunorubicin and idarubicin by reducing C13 ketonic group.

Daunorubicin, idarubicin, doxorubicin and epirubicin are anthracyclines widely used for the treatment of lymphoma, leukemia, and breast, lung, and liver cancers, but tumor resistance limits their clinical success. Aldo-keto reductase family 1 B10 (AKR1B10) is an NADPH-dependent enzyme overexpressed in liver and lung carcinomas. This study was aimed to determine the role of AKR1B10 in tumor resistance to anthracyclines. AKR1B10 activity toward anthracyclines was measured using recombinant protein. Cell resistance to anthracycline was determined by ectopic expression of AKR1B10 or inhibition by epalrestat. Results showed that AKR1B10 reduces C13-ketonic group on side chain of daunorubicin and idarubicin to hydroxyl forms. In vitro, AKR1B10 converted daunorubicin to daunorubicinol at V(max) of 837.42±81.39nmol/mg/min, K(m) of 9.317±2.25mM and k(cat)/K(m) of 3.24. AKR1B10 showed better catalytic efficiency toward idarubicin with V(max) at 460.23±28.12nmol/mg/min, K(m) at 0.461±0.09mM and k(cat)/K(m) at 35.94. AKR1B10 was less active toward doxorubicin and epirubicin with a C14-hydroxyl group. In living cells, AKR1B10 efficiently catalyzed reduction of daunorubicin (50nM) and idarubicin (30nM) to corresponding alcohols. Within 24h, approximately 20±2.7% of daunorubicin (1µM) or 23±2.3% of idarubicin (1µM) was converted to daunorubicinol or idarubicinol in AKR1B10 expression cells compared to 7±0.9% and 5±1.5% in vector control. AKR1B10 expression led to cell resistance to daunorubicin and idarubicin, but inhibitor epalrestat showed a synergistic role with these agents. Together our data suggest that AKR1B10 participates in cellular metabolism of daunorubicin and idarubicin, resulting in drug resistance. These data are informative for the clinical use of idarubicin and daunorubicin.

Connecting the dots on vertical transmission of SARS-CoV-2 using protein-protein interaction network analysis - Potential roles of placental ACE2 and ENDOU.

We conducted a protein-protein interaction (PPI) network study searching for proteins relevant to pregnancy-associated COVID-19 in pregnancy complicated with severe preeclampsia (sPE) and intra-amniotic infection and/or inflammation (Triple-I). PPI networks from sPE and Triple-I were intersected with the PPI network from coronavirus infection. Common proteins included the SARS-CoV-2 entry receptor ACE2 and ENDOU, a placental endoribonuclease homologous to Nsp15, a protein produced by the virus to escape host immunity. Remarkably, placental ENDOU mRNA expression far exceeded that of ACE2. Immunohistochemistry confirmed ENDOU localization at the hemochorial maternal-fetal interface. Investigation of ENDOU's relevance to vertical transmission of SARS-CoV-2 is further warranted.

Hydrogels derived from acellular porcine corneal stroma enhance corneal wound healing.

Acellular cornea derived hydrogels provide significant advantages in preserving native corneal stromal keratocyte cells and endothelial cells. However, for clinical application, hydrogel physical properties need to be improved, and their role in corneal epithelial wound healing requires further investigation. In this study, an acellular porcine corneal stroma (APCS) hydrogel (APCS-gel) was successfully prepared from 20 mg/ml APCS, demonstrated optimal light transmittance and gelation kinetic properties and retained critical corneal ECM of collagens and growth factors. Compared with fibrin gel, the APCS-gel had a higher porosity ratio and faster nutrition diffusion with an accompanying improvement in the proliferation of primary rabbit corneal epithelial cells (RCECs) and stromal cells (RCSCs). These corneal cell types also displayed improved viability and cellular infiltration. Furthermore, the APCS-gel provides significant advantages in the preservation of RCECs stemness and enhancement of corneal wound healing in vitro and in vivo. After 7 days of culture, 3-4 layers of RCECs were formed on the APCS-gel in vitro, while only 1-2 layers were found on the fibrin gel. More corneal stem/progenitor cell phenotypes (K12-, p63+, ABCG2+) and proliferation phenotypes (Ki67+) were detected on the APCS-gel than fibrin gel. Using a corneal epithelial wound healing model, we also found faster reepithelization in corneas that received APCS-gel compared to fibrin gel. Additionally, our APCS-gel demonstrated better physical and biological properties when compared to Tisseel, a clinically used type of fibrin gel. In conclusion, our APCS-gel provided better corneal epithelial and stromal cell biocompatibility to fibrin gels and due to its transparency and faster gelation time could potentially be superior for clinical purposes. STATEMENT OF SIGNIFICANCE: Extracellular matrix (ECM) can be used to provide tissue specific physical microstructure and biochemical cues for tissue regeneration. Here, we produced an ECM hydrogel derived from acellular porcine cornea stroma (APCS-gel) that retained critical biological characteristics of the native tissue and provided significant transparency and fast gelation time. Our data demonstrated that the APCS-gel was superior to clinically used fibrin gel, as the APCS-gel showed high porosity and permeability, better corneal stromal keratocytes infiltration, increased cellular proliferation and retention of corneal epithelial cells stemness. The APCS-gel improved corneal wound healing in vitro and in vivo. This APCS-gel may have clinical utility for corneal diseases, and the more general approach used to make this hydrogel might be used in other tissues.

Psoralen Derivatives with Enhanced Potency.

Psoralen is a furocoumarin natural product that intercalates within DNA and forms covalent adducts when activated by ultraviolet radiation. It is well known that this property contributes to psoralen's clinical efficacy in several disease contexts, which include vitiligo, psoriasis, graft-versus-host disease and cutaneous T-cell lymphoma. Given the therapeutic relevance of psoralen and its derivatives, we attempted to synthesize psoralens with even greater potency. In this study, we report a library of 73 novel psoralens, the largest collection of its kind. When screened for the ability to reduce cell proliferation, we identified two derivatives even more cytotoxic than 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT), one of the most potent psoralens identified to date. Using MALDI-TOF MS, we studied the DNA adduct formation for a subset of novel psoralens and found that in most cases enhanced DNA binding correlated well with cytotoxicity. Generally, our most potent derivatives contain positively charged substituents, which we believe increase DNA affinity and enhance psoralen intercalation. Thus, we provide a rational approach to guide efforts toward further optimizing psoralens to fully capitalize on this drug class' therapeutic potential. Finally, the structure-activity insights we have gained shed light on several opportunities to study currently underappreciated aspects of psoralen's mechanism.

Male marmoset monkeys express an adrenal fetal zone at birth, but not a zona reticularis in adulthood.

Neonatal human males produce high levels of dehydroepiandrosterone (DHEA) and its sulfo-conjugated form (DS) that decline within a few months of birth, due to regression of the adrenal fetal zone (FZ). Adult male humans and rhesus monkeys produce C19 steroids in abundance from the adrenal zona reticularis (ZR). Male marmoset monkeys produce DS at birth, but unlike humans and rhesus monkeys, do not produce comparable amounts of DHEA and DS in adulthood. To determine whether male marmosets express a functional ZR in adulthood, we examined adult and neonatal male marmosets for the presence of a ZR and FZ, respectively. Exogenous ACTH failed to stimulate DHEA or DS in adults, and dexamethasone treatment failed to suppress DHEA and DS, although cortisol levels changed as expected. In steroidogenic tissues, the key proteins necessary to synthesize C19 steroids from pregnenolone are P450c17, 3beta-hydroxysteroid dehydrogenase (3beta-HSD), nicotinamide adenine dinucleotide phosphate (reduced) oxido-reductase cytochrome P450 (reductase), and cytochromeb5 (cytb5). Adult adrenal cross sections showed P450c17 and reductase protein expression throughout the cortex but showed no expected decrease in 3beta-HSD and increase in cytb5 in the innermost region. Western analysis confirmed these data, demonstrating comparable P450c17 expression to rhesus monkeys, but not cytb5. HPLC analysis revealed similar 17alpha-hydroxylase action on pregnenolone for adult marmoset and rhesus adrenal microsomes but greatly diminished 17,20-lyase activity in marmosets. Neonatal marmoset adrenals exhibited staining indicative of a putative FZ (with P450c17, reduced 3beta-HSD and increased cytb5). We conclude that neonatal marmosets exhibit a C19 steroid-secreting FZ similar to humans, but adult males fail to acquire a functional ZR.

Determination of phthalate ester congeners and mixtures by LC/ESI-MS in sediments and biota of an urbanized marine inlet.

Phthalate esters (PEs) are a group of widely used commercial chemicals consisting of many different congeners. Concentrations of di(2-ethylhexyl) phthalate ester in the parts per million range have been observed in sediments from locations in North America and Europe. However, sediment and biota concentrations of other widely used PEs (i.e., dibutyl phthalate, diisononyl phthalate, and diisodecyl phthalate) are rare and often in doubt because of analytical difficulties. One of the problems is that commercial formulations predominantly consist of PEs with a specific molecular weight but include many isomers within each molecular weight class. Currently there are no analytical methods or required standards to fully separate PEs into the different molecular weight classes corresponding to the formulations from which they originate. Hence, ambient total and mixture-specific PE concentrations do not exist. This study presents a new method based on reversed-phase liquid chromatography/ electrospray ionization mass spectrometry (LC/ESI-MS) for the quantitative determination of individual PEs, including six congeners on the U.S. EPA Priority pollutant list and several commercial PE isomeric mixtures, in complex environmental matrixes. The method is applied to determine the composition of PE concentrations in sediments and fish in an urbanized marine ecosystem. PE fingerprints in sediments show a predominance of high molecular weight PEs and match per capita consumption levels of PEs. Fingerprints in fish tissue show a predominance of low molecular weight PEs and do not match per capita consumption levels. The findings indicate that the higher molecular weight PEs are less biologically available than the lower molecular weight ones.