Comparison of Serum and Cervical Cytokine Levels Throughout Pregnancy Between Preterm and Term Births

Objective: To assess differences in cytokine levels in cervicovaginal fluid (CVF) and serum across trimesters between women with preterm births (PTBs) and full-term births. Study Design: This multicenter study enrolled 302 women with a singleton gestation. CVF and serum cytokines, interleukin 1α (IL-1α), IL-1β, IL-6, IL-8, IL-10, C-reactive protein (CRP), tumor necrosis factor (TNF)-α, and matrix metalloproteinase (MMP)-8, were measured. Women with at least one cytokine assessment and noted PTB status in their medical record were retained in the study (N ¼ 272). Data were analyzed using mixed modeling (main effects of PTBs and time/trimester). Results: For the CVF values of IL-6, IL-8, IL-10, TNF-α, and CRP, and serum MMP-8, those who delivered preterm had significantly higher values than the full-term group regardless of trimester. For the serum values of IL-1β, IL-6, and TNF-α, those delivering preterm had significantly lower values than those delivering full-term regardless of trimester. For IL-1β in CVF, the cytokine was significantly higher in the PTB group for second and third trimesters only, relative to the full-term group. Conclusion: For each CVF cytokine that differed by birth status, values were higher for PTB than term, averaged over trimester. Numerous cytokine profiles varied across trimesters in women delivering term versus preterm in both CVF and serum

Comparison of Serum and Cervical Cytokine Levels Throughout Pregnancy Between Preterm and Term Births

Objective To assess differences in cytokine levels in cervicovaginal fluid (CVF) and serum across trimesters between women with preterm births (PTBs) and full-term births. Study Design This multicenter study enrolled 302 women with a singleton gestation. CVF and serum cytokines, interleukin 1α (IL-1α), IL-1β, IL-6, IL-8, IL-10, C-reactive protein (CRP), tumor necrosis factor (TNF)-α, and matrix metalloproteinase (MMP)-8, were measured. Women with at least one cytokine assessment and noted PTB status in their medical record were retained in the study (N = 272). Data were analyzed using mixed modeling (main effects of PTBs and time/trimester). Results For the CVF values of IL-6, IL-8, IL-10, TNF-α, and CRP, and serum MMP-8, those who delivered preterm had significantly higher values than the full-term group regardless of trimester. For the serum values of IL-1β, IL-6, and TNF-α, those delivering preterm had significantly lower values than those delivering full-term regardless of trimester. For IL-1β in CVF, the cytokine was significantly higher in the PTB group for second and third trimesters only, relative to the full-term group. Conclusion For each CVF cytokine that differed by birth status, values were higher for PTB than term, averaged over trimester. Numerous cytokine profiles varied across trimesters in women delivering term versus preterm in both CVF and serum

Computational study of substrate isotope effect probes of transition state structure for acetylcholinesterase catalysis

Secondary isotope effects for carbonyl addition reactions of methyl thioacetate, acetone and acetaldehyde have been calculated by ab initio quantum mechanical methods in an effort to interpret measured beta-deuterium isotope effects on acetylcholinesterase- catalyzed hydrolysis of acetylthiocholine. The calculated beta-deuterium isotope effect for equilibrium addition of methanol to methyl thioacetate is D3Keq = 0.965, and the corresponding effect for addition of methoxide ion to methyl thioacetate wherein three waters are hydrogen bonded to the carbonyl oxyanion is D3Keq = 1.086. Neither of these calculated isotope effects is as inverse as the experimental beta-deuterium isotope effect for acetylcholinesterase-catalyzed hydrolysis of acetylthiocholine, D3kE = 0.90š0.03. Structural comparisons show that the water-solvated methoxide adduct of methyl thioacetate is more expanded than is the neutral methanol addition adduct, and suggest that the degree to which the isotope effect is inverse (i.e. less than) is inversely correlated to the degree of expansion of the adduct. A similar correlation of beta-deuterium and beta-deuterium secondary isotope effects with the degree of expansion of the adducts is found for equilibrium additions of methanol and methoxide ion to acetaldehyde. These computational results suggest that the markedly inverse beta-deuterium isotope effect for the acetylcholinesterase reaction arises from enzymic compression of the transition state

A nonalcoholic fatty liver disease model in human induced pluripotent stem cell-derived hepatocytes, created by endoplasmic reticulum stress-induced steatosis

Hepatic steatosis, a reversible state of metabolic dysregulation, can promote the onset of nonalcoholic steatohepatitis (NASH), and its transition is thought to be critical in disease evolution. The association between endoplasmic reticulum (ER) stress response and hepatocyte metabolism disorders prompted us to characterize ER stress-induced hepatic metabolic dysfunction in human induced pluripotent stem cell-derived hepatocytes (hiPSC-Hep), to explore regulatory pathways and validate a phenotypic in vitro model for progression of liver steatosis. We treated hiPSC-Hep with a ratio of unsaturated and saturated fatty acids in the presence of an inducer of ER stress to synergistically promote triglyceride accumulation and dysregulate lipid metabolism. We monitored lipid accumulation by high-content imaging and measured gene regulation by RNA sequencing and reverse transcription quantitative PCR analyses. Our results show that ER stress potentiated intracellular lipid accumulation by 5-fold in hiPSC-Hep in the absence of apoptosis. Transcriptome pathway analysis identified ER stress pathways as the most significantly dysregulated of all pathways affected. Obeticholic acid dose dependently inhibited lipid accumulation and modulated gene expression downstream of the farnesoid X receptor. We were able to identify modulation of hepatic markers and gene pathways known to be involved in steatosis and nonalcoholic fatty liver disease (NAFLD), in support of a hiPSC-Hep disease model that is relevant to clinical data for human NASH. Our results show that the model can serve as a translational discovery platform for the understanding of molecular pathways involved in NAFLD, and can facilitate the identification of novel therapeutic molecules based on high-throughput screening strategies

Point mutations identify the glutamate binding pocket of the N-methyl-D-aspartate receptor as major site of conantokin-G inhibition.

Conantokin-G (Con-G), a gamma-carboxylglutamate (Gla) containing peptide derived from the venom of the marine cone snail Conus geographus, acts as a selective and potent inhibitor of N-methyl-D-aspartate (NMDA) receptors. Here, the effect of Con-G on recombinant NMDA receptors carrying point mutations within the glycine and glutamate binding pockets of the NR1 and NR2B subunits was studied using whole-cell voltage-clamp recording from cRNA injected Xenopus oocytes. At wild-type receptors, glutamate-induced currents were inhibited by Con-G in a dose-dependent manner at concentrations of 0.1-100 microM. Substitution of selected residues within the NR2B subunit reduced the inhibitory potency of Con-G, whereas similar mutations in the NR1 subunit had little effect. These results indicate a selective interaction of Con-G with the glutamate binding pocket of the NMDA receptor. Homology-based molecular modeling of the glutamate binding region based on the known structure of the glutamate binding site of the AMPA receptor protein GluR2 suggests how selected amino acid side chains of NR2B might interact with specific residues of Con-G

Human skeletal muscle tissue chip autonomous payload reveals changes in fiber type and metabolic gene expression due to spaceflight

Abstract Microphysiological systems provide the opportunity to model accelerated changes at the human tissue level in the extreme space environment. Spaceflight-induced muscle atrophy experienced by astronauts shares similar physiological changes to muscle wasting in older adults, known as sarcopenia. These shared attributes provide a rationale for investigating molecular changes in muscle cells exposed to spaceflight that may mimic the underlying pathophysiology of sarcopenia. We report the results from three-dimensional myobundles derived from muscle biopsies from young and older adults, integrated into an autonomous CubeLab™, and flown to the International Space Station (ISS) aboard SpaceX CRS-21 as part of the NIH/NASA funded Tissue Chips in Space program. Global transcriptomic RNA-Seq analyses comparing the myobundles in space and on the ground revealed downregulation of shared transcripts related to myoblast proliferation and muscle differentiation. The analyses also revealed downregulated differentially expressed gene pathways related to muscle metabolism unique to myobundles derived from the older cohort exposed to the space environment compared to ground controls. Gene classes related to inflammatory pathways were downregulated in flight samples cultured from the younger cohort compared to ground controls. Our muscle tissue chip platform provides an approach to studying the cell autonomous effects of spaceflight on muscle cell biology that may not be appreciated on the whole organ or organism level and sets the stage for continued data collection from muscle tissue chip experimentation in microgravity. We also report on the challenges and opportunities for conducting autonomous tissue-on-chip CubeLabTM payloads on the ISS

A chemical biology approach identified PI3K as a potential therapeutic target for neurofibromatosis type 2

Mutations in the merlin tumor suppressor gene cause Neurofibromatosis type 2 (NF2), which is a disease characterized by development of multiple benign tumors in the nervous system. The current standard of care for NF2 calls for surgical resection of the characteristic tumors, often with devastating neurological consequences. There are currently no approved non-surgical therapies for NF2. In an attempt to identify much needed targets and therapeutically active compounds for NF2 treatment, we employed a chemical biology approach using ultra-highthroughput screening. To support this goal, we created a merlin-null mouse Schwann cell (MSC) line to screen for compounds that selectively decrease their viability and proliferation. We optimized conditions for 384-well plate assays and executed a proof-of-concept screen of the Library of Pharmacologically Active Compounds. Further confirmatory and selectivity assays identified phosphatidylinositol 3-kinase (PI3K) as a potential NF2 drug target. Notably, loss of merlin function is associated with activation of the PI3K/Akt pathway in human schwannomas. We report that AS605240, a PI3K inhibitor, decreased merlin-null MSC viability in a dose-dependent manner without significantly decreasing viability of control Schwann cells. AS605240 exerted its action on merlin-null MSCs by promoting caspase-dependent apoptosis and inducing autophagy. Additional PI3K inhibitors tested also decreased viability of merlin-null MSCs in a dose-dependent manner. In summary, our chemical genomic screen and subsequent hit validation studies have identified PI3K as potential target for NF2 therapy

Discovery of ML358, a Selective Small Molecule Inhibitor of the SKN‑1 Pathway Involved in Drug Detoxification and Resistance in Nematodes

Nematodes parasitize ∼1/3 of humans worldwide, and effective treatment via administration of anthelmintics is threatened by growing resistance to current therapies. The nematode transcription factor SKN-1 is essential for development of embryos and upregulates the expression of genes that result in modification, conjugation, and export of xenobiotics, which can promote resistance. Distinct differences in regulation and DNA binding relative to mammalian Nrf2 make SKN-1 a promising and selective target for the development of anthelmintics with a novel mode of action that targets stress resistance and drug detoxification. We report <b>17</b> (<b>ML358</b>), a first in class small molecule inhibitor of the SKN-1 pathway. Compound <b>17</b> resulted from a vanillamine-derived hit identified by high throughput screening that was advanced through analog synthesis and structure–activity studies. Compound <b>17</b> is a potent (IC₅₀ = 0.24 μM, <i>E</i>_max = 100%) and selective inhibitor of the SKN-1 pathway and sensitizes the model nematode <i>C. elegans</i> to oxidants and anthelmintics. Compound <b>17</b> is inactive against Nrf2, the homologous mammalian detoxification pathway, and is not toxic to <i>C. elegans</i> (LC₅₀ > 64 μM) and Fa2N-4 immortalized human hepatocytes (LC₅₀ > 5.0 μM). In addition, <b>17</b> exhibits good solubility, permeability, and chemical and metabolic stability in human and mouse liver microsomes. Therefore, <b>17</b> is a valuable probe to study regulation and function of SKN-1 <i>in vivo</i>. By selective targeting of the SKN-1 pathway, <b>17</b> could potentially lead to drug candidates that may be used as adjuvants to increase the efficacy and useful life of current anthelmintics

Design of High-Throughput Screening Assays and Identification of a SUMO1-Specific Small Molecule Chemotype Targeting the SUMO-Interacting Motif-Binding Surface

Protein–protein interactions are generally challenging to target by small molecules. To address the challenge, we have used a multidisciplinary approach to identify small-molecule disruptors of protein–protein interactions that are mediated by SUMO (small ubiquitin-like modifier) proteins. SUMO modifications have emerged as a target with importance in treating cancer, neurodegenerative disorders, and viral infections. It has been shown that inhibiting SUMO-mediated protein–protein interactions can sensitize cancer cells to chemotherapy and radiation. We have developed highly sensitive assays using time-resolved fluorescence resonance energy transfer (TR-FRET) and fluorescence polarization (FP) that were used for high-throughput screening (HTS) to identify inhibitors for SUMO-dependent protein–protein interactions. Using these assays, we have identified a nonpeptidomimetic small molecule chemotype that binds to SUMO1 but not SUMO2 or 3. NMR chemical shift perturbation studies have shown that the compounds of this chemotype bind to the SUMO1 surface required for protein–protein interaction, despite the high sequence similarity of SUMO1 and SUMO2 and 3 at this surface