Cell trajectory modeling identifies a primitive trophoblast state defined by BCAM enrichment.

In early placental development, progenitor cytotrophoblasts (CTB) differentiate along one of two cellular trajectories: the villous or extravillous pathways. CTB committed to the villous pathway fuse with neighboring CTB to form the outer multinucleated syncytiotrophoblast (SCT), whereas CTB committed to the extravillous pathway differentiate into invasive extravillous trophoblasts (EVT). Unfortunately, little is known about the processes controlling human CTB progenitor maintenance and differentiation. To address this, we established a single cell RNA sequencing (scRNA-seq) dataset from first trimester placentas to identify cell states important in trophoblast progenitor establishment, renewal and differentiation. Multiple distinct trophoblast states were identified, representing progenitor CTB, column CTB, SCT precursors and EVT. Lineage trajectory analysis identified a progenitor origin that was reproduced in human trophoblast stem cell organoids. Heightened expression of basal cell adhesion molecule (BCAM) defined this primitive state, where BCAM enrichment or gene silencing resulted in enhanced or diminished organoid growth, respectively. Together, this work describes at high-resolution trophoblast heterogeneity within the first trimester, resolves gene networks within human CTB progenitors and identifies BCAM as a primitive progenitor marker and possible regulator.

Probing Watson-Crick and Hoogsteen base pairing in duplex DNA using dynamic nuclear polarization solid-state NMR spectroscopy.

The majority of base pairs in double-stranded DNA exist in the canonical Watson-Crick geometry. However, they can also adopt alternate Hoogsteen conformations in various complexes of DNA with proteins and small molecules, which are key for biological function and mechanism. While detection of Hoogsteen base pairs in large DNA complexes and assemblies poses considerable challenges for traditional structural biology techniques, we show here that multidimensional dynamic nuclear polarization-enhanced solid-state NMR can serve as a unique spectroscopic tool for observing and distinguishing Watson-Crick and Hoogsteen base pairs in a broad range of DNA systems based on characteristic NMR chemical shifts and internuclear dipolar couplings. We illustrate this approach using a model 12-mer DNA duplex, free and in complex with the antibiotic echinomycin, which features two central adenine-thymine base pairs with Watson-Crick and Hoogsteen geometry, respectively, and subsequently extend it to the ∼200 kDa Widom 601 DNA nucleosome core particle.

Conformational and Interaction Landscape of Histone H4 Tails in Nucleosomes Probed by Paramagnetic NMR Spectroscopy.

The fundamental repeat unit of chromatin, the nucleosome, consists of approximately 147 base pairs of double-stranded DNA and a histone protein octamer containing two copies each of histones H2A, H2B, H3, and H4. Each histone possesses a dynamically disordered N-terminal tail domain, and it is well-established that the tails of histones H3 and H4 play key roles in chromatin compaction and regulation. Here we investigate the conformational ensemble and interactions of the H4 tail in nucleosomes by means of solution NMR measurements of paramagnetic relaxation enhancements (PREs) in recombinant samples reconstituted with 15N-enriched H4 and nitroxide spin-label tagged H3. The experimental PREs, which report on the proximities of individual H4 tail residues to the different H3 spin-label sites, are interpreted by using microsecond time-scale molecular dynamics simulations of the nucleosome core particle. Collectively, these data enable improved localization of histone H4 tails in nucleosomes and support the notion that H4 tails engage in a fuzzy complex interaction with nucleosomal DNA.

Histone H4 Tails in Nucleosomes: a Fuzzy Interaction with DNA.

The interaction of positively charged N-terminal histone tails with nucleosomal DNA plays an important role in chromatin assembly and regulation, modulating their susceptibility to post-translational modifications and recognition by chromatin-binding proteins. Here, we report residue-specific 15 N NMR relaxation rates for histone H4 tails in reconstituted nucleosomes. These data indicate that H4 tails are strongly dynamically disordered, albeit with reduced conformational flexibility compared to a free peptide with the same sequence. Remarkably, the NMR observables were successfully reproduced in a 2-µs MD trajectory of the nucleosome. This is an important step toward resolving an apparent inconsistency where prior simulations were generally at odds with experimental evidence on conformational dynamics of histone tails. Our findings indicate that histone H4 tails engage in a fuzzy interaction with nucleosomal DNA, underpinned by a variable pattern of short-lived salt bridges and hydrogen bonds, which persists at low ionic strength (0-100 mM NaCl).

Conformational Dynamics in the Core of Human Y145Stop Prion Protein Amyloid Probed by Relaxation Dispersion NMR.

Microsecond to millisecond timescale backbone dynamics of the amyloid core residues in Y145Stop human prion protein (PrP) fibrils were investigated by using 15 N rotating frame (R1ρ ) relaxation dispersion solid-state nuclear magnetic resonance spectroscopy over a wide range of spin-lock fields. Numerical simulations enabled the experimental relaxation dispersion profiles for most of the fibril core residues to be modelled by using a two-state exchange process with a common exchange rate of 1000 s-1 , corresponding to protein backbone motion on the timescale of 1 ms, and an excited-state population of 2 %. We also found that the relaxation dispersion profiles for several amino acids positioned near the edges of the most structured regions of the amyloid core were better modelled by assuming somewhat higher excited-state populations (∼5-15 %) and faster exchange rate constants, corresponding to protein backbone motions on the timescale of ∼100-300 µs. The slow backbone dynamics of the core residues were evaluated in the context of the structural model of human Y145Stop PrP amyloid.

Understanding Carbon Dioxide Solubility in Ionic Liquids by Exploring the Link with Liquid Clathrate Formation.

The solubilities of the quadrupolar molecules benzene and CO2 in various ionic liquids (ILs) are compared in order to determine the connection between aromatic liquid clathrate formation and CO2 dissolution in ILs. It was found that both CO2 Henry's law constants and benzene solubility are remarkably well correlated with each other and with IL molar volume, suggesting both phenomena depend more on the strength of interionic interactions between the ions of an IL rather than the identity of either ion. However, IL ion-quadrupole interactions were found to have an effect for dicyanamide ([N(CN)2 ]- ), where solubility of CO2 and benzene are affected by destabilizing and stabilizing interactions with [N(CN)2 ]- , respectively. The results suggest both solubility phenomena are related to the incorporation of the solute into an IL host network. Aromatic liquid clathrate formation thus has potential as a facile experimental probe for predicting the relative ability of ILs to physisorb CO2 .

Single-cell assessment of primary and stem cell-derived human trophoblast organoids as placenta-modeling platforms.

Human trophoblast stem cells (hTSCs) and related trophoblast organoids are state-of-the-art culture systems that facilitate the study of trophoblast development and human placentation. Using single-cell transcriptomics, we evaluate how organoids derived from freshly isolated first-trimester trophoblasts or from established hTSC cell lines reproduce developmental cell trajectories and transcriptional regulatory processes defined in vivo. Although organoids from primary trophoblasts and hTSCs overall model trophoblast differentiation with accuracy, specific features related to trophoblast composition, trophoblast differentiation, and transcriptional drivers of trophoblast development show levels of misalignment. This is best illustrated by the identification of an expanded progenitor state in stem cell-derived organoids that is nearly absent in vivo and transcriptionally shares both villous cytotrophoblast and extravillous trophoblast characteristics. Together, this work provides a comprehensive resource that identifies strengths and limitations of current trophoblast organoid platforms.

Global dot integration in typically developing children and in Williams syndrome.

Williams Syndrome (WS) is a neurodevelopmental disorder that results in deficits in visuospatial perception and cognition. The dorsal stream vulnerability hypothesis in WS predicts that visual motion processes are more susceptible to damage than visual form processes. We asked WS participants and typically developing children to detect the global structure Glass patterns, under "static" and "dynamic" conditions in order to evaluate this hypothesis. Sequentially presented Glass patterns are coined as dynamic because they induce illusory motion, which is modeled after the interaction between orientation (form) and direction (motion) mechanisms. If the dorsal stream vulnerability holds in WS participants, then they should process real and illusory motion atypically. However, results are consistent with the idea that form and motion integration mechanisms are functionally delayed or attenuated in WS. Form coherence thresholds for both static and dynamic Glass patterns in WS were similar to those of 4-5year old children, younger than what is predicted by mental age. Dynamic presentation of Glass patterns improved thresholds to the same degree as typical participants. Motion coherence thresholds in WS were similar to those of mental age matches. These data pose constraints on the dorsal vulnerability hypothesis, and refine our understanding of the relationship between form and motion processing in development.

Plasma Environment, Radiation, Structure, and Evolution of the Uranian System (PERSEUS): A Dedicated Orbiter Mission Concept to Study Space Physics at Uranus.

The Plasma Environment, Radiation, Structure, and Evolution of the Uranian System (PERSEUS) mission concept defines the feasibility and potential scope of a dedicated, standalone Heliophysics orbiter mission to study multiple space physics science objectives at Uranus. Uranus's complex and dynamic magnetosphere presents a unique laboratory to study magnetospheric physics as well as its coupling to the solar wind and the planet's atmosphere, satellites, and rings. From the planet's tilted and offset, rapidly-rotating non-dipolar magnetic field to its seasonally-extreme interactions with the solar wind to its unexpectedly intense electron radiation belts, Uranus hosts a range of outstanding and compelling mysteries relevant to the space physics community. While the exploration of planets other than Earth has largely fallen within the purview of NASA's Planetary Science Division, many targets, like Uranus, also hold immense scientific value and interest to NASA's Heliophysics Division. Exploring and understanding Uranus's magnetosphere is critical to make fundamental gains in magnetospheric physics and the understanding of potential exoplanetary systems and to test the validity of our knowledge of magnetospheric dynamics, moon-magnetosphere interactions, magnetosphere-ionosphere coupling, and solar wind-planetary coupling. The PERSEUS mission concept study, currently at Concept Maturity Level (CML) 4, comprises a feasible payload that provides closure to a range of space physics science objectives in a reliable and mature spacecraft and mission design architecture. The mission is able to close using only a single Mod-1 Next-Generation Radioisotope Thermoelectric Generator (NG-RTG) by leveraging a concept of operations that relies of a significant hibernation mode for a large portion of its 22-day orbit.

Single Live Cell Imaging of Multidrug Resistance Using Silver Ultrasmall Nanoparticles as Biosensing Probes in Triple-Negative Breast Cancer Cells.

Silver ultrasmall nanoparticles (Ag UNPs) (size < 5 nm) were used as biosensing probes to analyze the efflux kinetics contributing to multidrug resistance (MDR) in single live triple-negative breast cancer (TNBC) cells by using dark-field optical microscopy to follow their size-dependent localized surface plasmon resonance. TNBC cells lack expression of estrogen (ER-), progesterone (PR-), and human epidermal growth factor 2 (HER2-) receptors and are more likely to acquire resistance to anticancer drugs due to their ability to transport harmful substances outside the cell. The TNBC cells displayed greater nuclear and cytoplasmic efflux, resulting in less toxicity of Ag UNPs in a concentration-independent manner. In contrast, more Ag UNPs and an increase in cytotoxic effects were observed in the receptor-positive breast cancer cells that have receptors for ER+, PR+, and HER2+ and are known to better respond to anticancer therapies. Ag UNPs accumulated in receptor-positive breast cancer cells in a time-and concentration-dependent mode and caused decreased cellular growth, whereas the TNBC cells due to the efflux were able to continue to grow. The TNBC cells demonstrated a marked increase in survival due to their ability to have MDR determined by efflux of Ag UNPs outside the nucleus and the cytoplasm of the cells. Further evaluation of the nuclear efflux kinetics of TNBC cells with Ag UNPs as biosensing probes is critical to gain a better understanding of MDR and potential for enhancement of cancer drug delivery.

Transcriptomic mapping of the metzincin landscape in human trophoblasts.

The metzincin family of metalloproteases coordinates tissue developmental processes through regulation of growth factor availability, receptor signaling, and cell-cell/cell-matrix adhesion. While roles for select metzincins in controlling trophoblast functions in human placental development have been described, a comprehensive understanding of metzincin dynamics during trophoblast differentiation is lacking. To address this knowledge gap, single cell transcriptomic datasets derived from first trimester chorionic villi and decidua were used to decipher metzincin expression profiles and kinetics in diverse cell types within the utero-placental interface. Further, specific protease-substrate interactions within progenitor trophoblasts were examined to better define the progenitor niche. Within the uterine-placental compartment, 43 metzincin proteases were expressed across 15 cell-type clusters. Metzincin subgroups expressed in placental trophoblasts, placental mesenchymal cells, uterine stromal, and immune cells included multiple matrix metalloproteases (MMPs), a disintegrin and metalloproteases (ADAMs), a disintegrin and metalloproteases with thrombospondin repeats (ADAMTSs), pappalysins, and astacins. Within the trophoblast compartment, eight distinct trophoblasts states were identified: four cytotrophoblast (CTB), one syncytiotrophoblast precursor (SCTp), two column CTB (cCTB), and one extravillous trophoblast (EVT). Within these states 7 MMP, 8 ADAM, 4 ADAMTS, 2 pappalysin, and 3 astacin proteases were expressed. Cell trajectory modeling shows that expression of most (19/24) metzincins increase during EVT differentiation, though expression of select metalloproteases increase along the villous pathway. Eleven metzincins (ADAM10, -17, MMP14, -15, -19, -23B, ADAMTS1, -6, -19, TLL-1, -2) showed enrichment within CTB progenitors, and analysis of metzincin-substrate interactions identified ∼150 substrates and binding partners, including FBN2 as an ADAMTS6-specific substrate. Together, this work characterizes the metzincin landscape in human first trimester trophoblasts and establishes insight into the roles specific proteases perform within distinct trophoblast niches and across trophoblast differentiation. This resource serves as a guide for future investigations into the roles of metzincin proteases in human placental development.

Conformational Dynamics of Histone H3 Tails in Chromatin.

Chromatin is a supramolecular DNA-protein complex that compacts eukaryotic genomes and regulates their accessibility and functions. Dynamically disordered histone H3 N-terminal tails are among key chromatin regulatory components. Here, we used high-resolution-magic-angle-spinning NMR measurements of backbone amide 15N spin relaxation rates to investigate, with residue-specific detail, the dynamics and interactions of H3 tails in recombinant 13C,15N-enriched nucleosome arrays containing 15, 30, or 60 bp linker DNA between the nucleosome repeats. These measurements were compared to analogous data available for mononucleosomes devoid of linker DNA or containing two 20 bp DNA overhangs. The H3 tail dynamics in nucleosome arrays were found to be considerably attenuated compared with nucleosomes with or without linker DNA due to transient electrostatic interactions with the linker DNA segments and the structured chromatin environment. Remarkably, however, the H3 tail dynamics were not modulated by the specific linker DNA length within the 15-60 bp range investigated here.

Imprinted Gene Expression and Function of the Dopa Decarboxylase Gene in the Developing Heart.

Dopa decarboxylase (DDC) synthesizes serotonin in the developing mouse heart where it is encoded by Ddc_exon1a, a tissue-specific paternally expressed imprinted gene. Ddc_exon1a shares an imprinting control region (ICR) with the imprinted, maternally expressed (outside of the central nervous system) Grb10 gene on mouse chromosome 11, but little else is known about the tissue-specific imprinted expression of Ddc_exon1a. Fluorescent immunostaining localizes DDC to the developing myocardium in the pre-natal mouse heart, in a region susceptible to abnormal development and implicated in congenital heart defects in human. Ddc_exon1a and Grb10 are not co-expressed in heart nor in brain where Grb10 is also paternally expressed, despite sharing an ICR, indicating they are mechanistically linked by their shared ICR but not by Grb10 gene expression. Evidence from a Ddc_exon1a gene knockout mouse model suggests that it mediates the growth of the developing myocardium and a thinning of the myocardium is observed in a small number of mutant mice examined, with changes in gene expression detected by microarray analysis. Comparative studies in the human developing heart reveal a paternal expression bias with polymorphic imprinting patterns between individual human hearts at DDC_EXON1a, a finding consistent with other imprinted genes in human.

EPHB6 augments both development and drug sensitivity of triple-negative breast cancer tumours.

Triple-negative breast cancer (TNBC) tumours that lack expression of oestrogen, and progesterone receptors, and do not overexpress the HER2 receptor represent the most aggressive breast cancer subtype, which is characterised by the resistance to therapy in frequently relapsing tumours and a high rate of patient mortality. This is likely due to the resistance of slowly proliferating tumour-initiating cells (TICs), and understanding molecular mechanisms that control TICs behaviour is crucial for the development of effective therapeutic approaches. Here, we present our novel findings, indicating that an intrinsically catalytically inactive member of the Eph group of receptor tyrosine kinases, EPHB6, partially suppresses the epithelial-mesenchymal transition in TNBC cells, while also promoting expansion of TICs. Our work reveals that EPHB6 interacts with the GRB2 adapter protein and that its effect on enhancing cell proliferation is mediated by the activation of the RAS-ERK pathway, which allows it to elevate the expression of the TIC-related transcription factor, OCT4. Consistent with this, suppression of either ERK or OCT4 activities blocks EPHB6-induced pro-proliferative responses. In line with its ability to trigger propagation of TICs, EPHB6 accelerates tumour growth, potentiates tumour initiation and increases TIC populations in xenograft models of TNBC. Remarkably, EPHB6 also suppresses tumour drug resistance to DNA-damaging therapy, probably by forcing TICs into a more proliferative, drug-sensitive state. In agreement, patients with higher EPHB6 expression in their tumours have a better chance for recurrence-free survival. These observations describe an entirely new mechanism that governs TNBC and suggest that it may be beneficial to enhance EPHB6 action concurrent with applying a conventional DNA-damaging treatment, as it would decrease drug resistance and improve tumour elimination.

Rapid Quantitative Measurements of Paramagnetic Relaxation Enhancements in Cu(II)-Tagged Proteins by Proton-Detected Solid-State NMR Spectroscopy.

We demonstrate rapid quantitative measurements of site-resolved paramagnetic relaxation enhancements (PREs), which are a source of valuable structural restraints corresponding to electron-nucleus distances in the ∼10-20 Å regime, in solid-state nuclear magnetic resonance (NMR) spectra of proteins containing covalent Cu2+-binding tags. Specifically, using protein GB1 K28C-EDTA-Cu2+ mutant as a model, we show the determination of backbone amide 15N longitudinal and 1H transverse PREs within a few hours of experiment time based on proton-detected 2D or 3D correlation spectra recorded with magic-angle spinning frequencies ≥ ∼ 60 kHz for samples containing ∼10-50 nanomoles of 2H,13C,15N-labeled protein back-exchanged in H2O. Additionally, we show that the electron relaxation time for the Cu2+ center, needed to convert PREs into distances, can be estimated directly from the experimental data. Altogether, these results are important for establishing solid-state NMR based on paramagnetic-tagging as a routine tool for structure determination of natively diamagnetic proteins.

Cysteine-specific Cu2+ chelating tags used as paramagnetic probes in double electron electron resonance.

Double electron electron resonance (DEER) is an attractive technique that is utilized for gaining insight into protein structure and dynamics via nanometer-scale distance measurements. The most commonly used paramagnetic tag in these measurements is a nitroxide spin label, R1. Here, we present the application of two types of high-affinity Cu(2+) chelating tags, based on the EDTA and cyclen metal-binding motifs as alternative X-band DEER probes, using the B1 immunoglobulin-binding domain of protein G (GB1) as a model system. Both types of tags have been incorporated into a variety of protein secondary structure environments and exhibit high spectral sensitivity. In particular, the cyclen-based tag displays distance distributions with comparable distribution widths and most probable distances within 1-3 Å when compared to homologous R1 distributions. The results display the viability of the cyclen tag as an alternative to the R1 side chain for X-band DEER distance measurements in proteins.

Poly(ionic liquid) superabsorbent for polar organic solvents.

A simple, polymerized ionic liquid (poly(IL)) based on methylimidazolium cations tethered to a polystyrene backbone exhibits superabsorbent behavior toward polar organic solvents, most notably propylene carbonate (PC) and dimethyl sulfoxide (DMSO), wherein the poly(IL) was observed to swell more than 390 and 200 times (w/w) its original mass, yet absorbs negligible quantities of water, hexanes, and other solvents, many of which were miscible with the IL monomer. Although solubility parameters and dielectric constants are typically used to rationalize such behaviors, we find that poly(IL)-solvent compatibility is most clearly correlated to solvent dipole moment. Poly(IL) superabsorbency is not reliant upon the addition of a cross-linking agent.

Nucleoside azide-alkyne cycloaddition reactions under solvothermal conditions or using copper vials in a ball mill.

Novel nucleoside analogues containing photoswitchable moieties were prepared using 'click' cycloaddition reactions between 5'-azido-5'-deoxythymidine and mono- or bis-N-propargylamide-substituted azobenzenes. In solution, high to quantitative yields were achieved using 5 mol% Cu(I) in the presence of a stabilizing ligand. 'Click' reactions using the monopropargylamides were also effected in the absence of added cuprous salts by the application of liquid assisted grinding (LAG) in metallic copper reaction vials. Specifically, high speed vibration ball milling (HSVBM) using a 3/32″ (2.38 mm) diameter copper ball (62 mg) at 60 Hz overnight in the presence of ethyl acetate lead to complete consumption of the 5'-azido nucleoside with clean conversion to the corresponding 1,3-triazole.

Detection of the Acetylcholinesterase Insecticide Resistance Mutation (G328A) in Natural Populations of Ceratitis capitata.

Wild Mediterranean fruit fly specimens collected from various regions worldwide were screened for the glycine to alanine (Gly->Ala) point mutation (G328A) in the acetylcholinesterase enzyme, presumably causing resistance to organophosphates. We found that the single nucleotide polymorphism (SNP) responsible for this amino acid change is located at the beginning of exon 6 of the Ccace2 gene. The identification of the exact location of the SNP permitted PCR primer design around this site and direct sequencing of the corresponding genomic region. We detected the resistance allele in natural Mediterranean fruit fly populations from Brazil and Spain, but not from other sites in four continents. The known treatment history of sites suggests that the resistance buildup is linked to organophosphate application in the field. The PCR-based detection provides a screening method useful for monitoring Mediterranean fruit fly insecticide resistance in local populations and improving pest management strategies accordingly.

Molecular simulation of the thermophysical properties of N-functionalized alkylimidazoles.

Molecular simulations are used to probe the thermophysical properties of a series of N-functionalized alkylimidazoles, ranging from N-methylimidazole to N-heptylimidazole. These compounds have been previously synthesized, and their solvation properties have been shown to be potentially useful for CO(2) capture from industrial sources. In this work, we use first-principles calculations to fit electrostatic charges to the molecular models, which are then used to perform a series of molecular dynamics simulations. Over a range of different temperatures, we benchmark the simulated densities and heat capacities against experimental measurements. Also, we predict the Henry's constants for CO(2) absorption and probe the solvents' structures using molecular simulation techniques, such as fractional free volume analysis and void distributions. We find that our simulations are able to closely reproduce the experimental benchmarks and add additional insight into the molecular structure of these fluids, with respect to their observed solvent properties.