Pursuing totipotency: authentic totipotent stem cells in culture.

Totipotent stem cells are transiently occurring in vivo cells that can form all cell types of the embryo including placenta, with their in vitro counterparts being actively pursued. Subsequently, totipotent-like cells are established with variable robustness and biological relevance. Here, we summarize current progress on capturing these cells in culture.

An RNAi screen of RNA helicases identifies eIF4A3 as a regulator of embryonic stem cell identity.

RNA helicases are involved in multiple steps of RNA metabolism to direct their roles in gene expression, yet their functions in pluripotency control remain largely unexplored. Starting from an RNA interference (RNAi) screen of RNA helicases, we identified that eIF4A3, a DEAD-box (Ddx) helicase component of the exon junction complex (EJC), is essential for the maintenance of embryonic stem cells (ESCs). Mechanistically, we show that eIF4A3 post-transcriptionally controls the pluripotency-related cell cycle regulators and that its depletion causes the loss of pluripotency via cell cycle dysregulation. Specifically, eIF4A3 is required for the efficient nuclear export of Ccnb1 mRNA, which encodes Cyclin B1, a key component of the pluripotency-promoting pathway during the cell cycle progression of ESCs. Our results reveal a previously unappreciated role for eIF4A3 and its associated EJC in maintaining stem cell pluripotency through post-transcriptional control of the cell cycle.

Directed Evolution of an Enhanced POU Reprogramming Factor for Cell Fate Engineering.

Transcription factor-driven cell fate engineering in pluripotency induction, transdifferentiation, and forward reprogramming requires efficiency, speed, and maturity for widespread adoption and clinical translation. Here, we used Oct4, Sox2, Klf4, and c-Myc driven pluripotency reprogramming to evaluate methods for enhancing and tailoring cell fate transitions, through directed evolution with iterative screening of pooled mutant libraries and phenotypic selection. We identified an artificially evolved and enhanced POU factor (ePOU) that substantially outperforms wild-type Oct4 in terms of reprogramming speed and efficiency. In contrast to Oct4, not only can ePOU induce pluripotency with Sox2 alone, but it can also do so in the absence of Sox2 in a three-factor ePOU/Klf4/c-Myc cocktail. Biochemical assays combined with genome-wide analyses showed that ePOU possesses a new preference to dimerize on palindromic DNA elements. Yet, the moderate capacity of Oct4 to function as a pioneer factor, its preference to bind octamer DNA and its capability to dimerize with Sox2 and Sox17 proteins remain unchanged in ePOU. Compared with Oct4, ePOU is thermodynamically stabilized and persists longer in reprogramming cells. In consequence, ePOU: 1) differentially activates several genes hitherto not implicated in reprogramming, 2) reveals an unappreciated role of thyrotropin-releasing hormone signaling, and 3) binds a distinct class of retrotransposons. Collectively, these features enable ePOU to accelerate the establishment of the pluripotency network. This demonstrates that the phenotypic selection of novel factor variants from mammalian cells with desired properties is key to advancing cell fate conversions with artificially evolved biomolecules.

IL10RA modulates crizotinib sensitivity in NPM1-ALK+ anaplastic large cell lymphoma.

Anaplastic large cell lymphoma (ALCL) is a T-cell malignancy predominantly driven by a hyperactive anaplastic lymphoma kinase (ALK) fusion protein. ALK inhibitors, such as crizotinib, provide alternatives to standard chemotherapy with reduced toxicity and side effects. Children with lymphomas driven by nucleophosmin 1 (NPM1)-ALK fusion proteins achieved an objective response rate to ALK inhibition therapy of 54% to 90% in clinical trials; however, a subset of patients progressed within the first 3 months of treatment. The mechanism for the development of ALK inhibitor resistance is unknown. Through genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) activation and knockout screens in ALCL cell lines, combined with RNA sequencing data derived from ALK inhibitor-relapsed patient tumors, we show that resistance to ALK inhibition by crizotinib in ALCL can be driven by aberrant upregulation of interleukin 10 receptor subunit alpha (IL10RA). Elevated IL10RA expression rewires the STAT3 signaling pathway, bypassing otherwise critical phosphorylation by NPM1-ALK. IL-10RA expression does not correlate with response to standard chemotherapy in pediatric patients, suggesting that a combination of crizotinib and chemotherapy could prevent ALK inhibitor resistance-specific relapse.

Probing the path for achieving a broad temperature plateau of the figure of merit in thermoelectric nanocomposite materials.

The efficiency of a thermoelectric device depends directly on the average figure of merit (zT) of the material. A high average zT requires a broad temperature plateau with a high zT, but state-of-the-art thermoelectric materials display a peaked zT over a narrow temperature range due to a strong temperature dependence of transport properties. In this work, using Boltzmann transport theory, we systematically investigate the underlying physics and propose a strategy for attaining a broad temperature plateau of zT through proper engineering of the interfacial barrier height in PbTe nanocomposite material. The optimized barrier height (U constantzT) not only enhances the zT but also maintains its high value over a wide temperature range [Tmin :Tmax ]. It has been found that for p = 2.8 × 1020 cm-3, the U constantzT is 0.112 eV at which zT varies between 1.9-2.14 over a wide temperature range of 550-850 K, resulting in a high average zT of 2.02 in comparison to a bulk value of 1.22. Also, for p = 5 × 1019 cm-3, UconstantzT is 0.102 eV at which zT varies between 1.046-1.435 for a temperature range of 300-600K, resulting in a high average zT of 1.27 over a bulk value of 0.844. The above results show that the range [Tmin :Tmax ] depends on carrier concentration which, in turn, determines the position of the Fermi level (Ef ) and Fermi window at Tmin and Tmax . To obtain a broad temperature plateau of zT, the findings show that at Tmin, Ef should lie inside the band and zT should show strong variation with barrier height, whereas at Tmax , Ef should lie in the band gap and zT should have little variation with barrier height. This trend allows us to choose UconstantzT which synergistically optimizes the transport properties at Tmin with Tmax to give a broad temperature plateau of zT. This work proposes a new advantage of interfacial scattering which enhances the average zT and also provides necessary guidelines to experimentalists for synthesizing a highly efficient thermoelectric device.

DNA-mediated dimerization on a compact sequence signature controls enhancer engagement and regulation by FOXA1.

FOXA1 is a transcription factor capable to bind silenced chromatin to direct context-dependent cell fate conversion. Here, we demonstrate that a compact palindromic DNA element (termed 'DIV' for its diverging half-sites) induces the homodimerization of FOXA1 with strongly positive cooperativity. Alternative structural models are consistent with either an indirect DNA-mediated cooperativity or a direct protein-protein interaction. The cooperative homodimer formation is strictly constrained by precise half-site spacing. Re-analysis of chromatin immunoprecipitation sequencing data indicates that the DIV is effectively targeted by FOXA1 in the context of chromatin. Reporter assays show that FOXA1-dependent transcriptional activity declines when homodimeric binding is disrupted. In response to phosphatidylinositol-3 kinase inhibition DIV sites pre-bound by FOXA1 such as at the PVT1/MYC locus exhibit a strong increase in accessibility suggesting a role of the DIV configuration in the chromatin closed-open dynamics. Moreover, several disease-associated single nucleotide polymorphisms map to DIV elements and show allelic differences in FOXA1 homodimerization, reporter gene expression and are annotated as quantitative trait loci. This includes the rs541455835 variant at the MAPT locus encoding the Tau protein associated with Parkinson's disease. Collectively, the DIV guides chromatin engagement and regulation by FOXA1 and its perturbation could be linked to disease etiologies.

Ionic Assembly, Anion-π, Magnetic, and Electronic Attributes of Ambient Stable Naphthalenediimide Radical Ions.

Organic spin-based molecular materials are considered to be attractive for the generation of functional materials with emergent optoelectronic, magnetic, or magneto-conductive properties. However, the major limitations to the utilization of organic spin-based systems are their high reactivity, instability, and propensity for dimerization. Herein, we report the synthesis, characterization, and magnetic and electronic studies of three ambient stable radical ions (1 a.+ , 1 b.+ , and 1 c.+ ). The radical ions 1 b.+ and 1 c.+ with BPh4 - and BF4 - counter anions, respectively, were synthesized in excellent yields by means of anion metathesis of 1 a.+ with Br- as its counter anion. Notably, synthesis of 1 a.+ was achieved in an ecofriendly, solvent-free protocol. The radical ions were characterized by means of single-crystal X-ray diffraction studies, which revealed the discrete nature of the radical ions and extensive hydrogen-bonding interactions within the radical ions and with the counter anions. Thus, radical ions can be organized to form infinite supramolecular arrays using weak noncovalent interactions. In addition, the Br- , BF4 - , and BPh4 - anions formed diverse types of anion-π interactions with the naphthalene and imide rings of the radical ions. The radical ions were characterized by means of X-band electron paramagnetic resonance (EPR) spectroscopy in solution and in the solid state. Magnetic studies revealed their paramagnetic nature in the range of 10 to 300 K. The radical ions exhibited high resistivity approaching the gigaohm (GΩ) scale. In addition, the radical ions exhibited panchromism.

Changing POU dimerization preferences converts Oct6 into a pluripotency inducer.

The transcription factor Oct4 is a core component of molecular cocktails inducing pluripotent stem cells (iPSCs), while other members of the POU family cannot replace Oct4 with comparable efficiency. Rather, group III POU factors such as Oct6 induce neural lineages. Here, we sought to identify molecular features determining the differential DNA-binding and reprogramming activity of Oct4 and Oct6. In enhancers of pluripotency genes, Oct4 cooperates with Sox2 on heterodimeric SoxOct elements. By re-analyzing ChIP-Seq data and performing dimerization assays, we found that Oct6 homodimerizes on palindromic OctOct more cooperatively and more stably than Oct4. Using structural and biochemical analyses, we identified a single amino acid directing binding to the respective DNA elements. A change in this amino acid decreases the ability of Oct4 to generate iPSCs, while the reverse mutation in Oct6 does not augment its reprogramming activity. Yet, with two additional amino acid exchanges, Oct6 acquires the ability to generate iPSCs and maintain pluripotency. Together, we demonstrate that cell type-specific POU factor function is determined by select residues that affect DNA-dependent dimerization.

Diversity among POU transcription factors in chromatin recognition and cell fate reprogramming.

The POU (Pit-Oct-Unc) protein family is an evolutionary ancient group of transcription factors (TFs) that bind specific DNA sequences to direct gene expression programs. The fundamental importance of POU TFs to orchestrate embryonic development and to direct cellular fate decisions is well established, but the molecular basis for this activity is insufficiently understood. POU TFs possess a bipartite 'two-in-one' DNA binding domain consisting of two independently folding structural units connected by a poorly conserved and flexible linker. Therefore, they represent a paradigmatic example to study the molecular basis for the functional versatility of TFs. Their modular architecture endows POU TFs with the capacity to accommodate alternative composite DNA sequences by adopting different quaternary structures. Moreover, associations with partner proteins crucially influence the selection of their DNA binding sites. The plentitude of DNA binding modes confers the ability to POU TFs to regulate distinct genes in the context of different cellular environments. Likewise, different binding modes of POU proteins to DNA could trigger alternative regulatory responses in the context of different genomic locations of the same cell. Prominent POU TFs such as Oct4, Brn2, Oct6 and Brn4 are not only essential regulators of development but have also been successfully employed to reprogram somatic cells to pluripotency and neural lineages. Here we review biochemical, structural, genomic and cellular reprogramming studies to examine how the ability of POU TFs to select regulatory DNA, alone or with partner factors, is tied to their capacity to epigenetically remodel chromatin and drive specific regulatory programs that give cells their identities.

Sensitivity analysis of discharge patterns of subthalamic nucleus in the model of basal ganglia in Parkinson disease.

Parkinson disease alters the information patterns in movement related pathways in brain. Experimental results performed on rats show that the activity patterns changes from single spike activity to mixed burst mode in Parkinson disease. However the cause of this change in activity pattern is not yet completely understood. Subthalamic nucleus is one of the main nuclei involved in the origin of motor dysfunction in Parkinson disease. In this paper, a single compartment conductance based model is considered which focuses on subthalamic nucleus and synaptic input from globus pallidus (external). This model shows highly nonlinear behavior with respect to various intrinsic parameters. Behavior of model has been presented with the help of activity patterns generated in healthy and Parkinson condition. These patterns have been compared by calculating their correlation coefficient for different values of intrinsic parameters. Results display that the activity patterns are very sensitive to various intrinsic parameters and calcium shows some promising results which provide insights into the motor dysfunction.

Fifteen-minute consultation: structured approach to management of a child with recurrent croup.

Recurrent croup is a distinct clinical entity from viral croup. It is not a specific diagnosis and its presence should alert the clinician to explore the underlying cause. We discuss an evidence-based structured approach to management of a child with recurrent croup.

Phase-ordering kinetics of the asymmetric Coulomb glass model.

We present results for phase-ordering kinetics in the Coulomb glass (CG) model, which describes electrons on a lattice with unscreened Coulombic repulsion. The filling factor is denoted by K∈[0,1]. For a square lattice with K=0.5 (symmetric CG), the ground state is a checkerboard with alternating electrons and holes. In this paper, we focus on the asymmetric CG where K≲0.5, i.e., the ground state is checkerboard-like with excess holes distributed uniformly. There is no explicit quenched disorder in our system, though the Coulombic interaction gives rise to frustration. We find that the evolution morphology is in the same dynamical universality class as the ordering ferromagnet. Further, the domain growth law is slightly slower than the Lifshitz-Cahn-Allen law, L(t)∼t^{1/2}, i.e., the growth exponent is underestimated. We speculate that this could be a signature of logarithmic growth in the asymptotic regime.

ZFP281 controls transcriptional and epigenetic changes promoting mouse pluripotent state transitions via DNMT3 and TET1.

The progression from naive through formative to primed in vitro pluripotent stem cell states recapitulates epiblast development in vivo during the peri-implantation period of mouse embryo development. Activation of the de novo DNA methyltransferases and reorganization of transcriptional and epigenetic landscapes are key events that occur during these pluripotent state transitions. However, the upstream regulators that coordinate these events are relatively underexplored. Here, using Zfp281 knockout mouse and degron knockin cell models, we identify the direct transcriptional activation of Dnmt3a/3b by ZFP281 in pluripotent stem cells. Chromatin co-occupancy of ZFP281 and DNA hydroxylase TET1, which is dependent on the formation of R-loops in ZFP281-targeted gene promoters, undergoes a "high-low-high" bimodal pattern regulating dynamic DNA methylation and gene expression during the naive-formative-primed transitions. ZFP281 also safeguards DNA methylation in maintaining primed pluripotency. Our study demonstrates a previously unappreciated role for ZFP281 in coordinating DNMT3A/3B and TET1 functions to promote pluripotent state transitions.

Highly cooperative chimeric super-SOX induces naive pluripotency across species.

Our understanding of pluripotency remains limited: iPSC generation has only been established for a few model species, pluripotent stem cell lines exhibit inconsistent developmental potential, and germline transmission has only been demonstrated for mice and rats. By swapping structural elements between Sox2 and Sox17, we built a chimeric super-SOX factor, Sox2-17, that enhanced iPSC generation in five tested species: mouse, human, cynomolgus monkey, cow, and pig. A swap of alanine to valine at the interface between Sox2 and Oct4 delivered a gain of function by stabilizing Sox2/Oct4 dimerization on DNA, enabling generation of high-quality OSKM iPSCs capable of supporting the development of healthy all-iPSC mice. Sox2/Oct4 dimerization emerged as the core driver of naive pluripotency with its levels diminished upon priming. Transient overexpression of the SK cocktail (Sox+Klf4) restored the dimerization and boosted the developmental potential of pluripotent stem cells across species, providing a universal method for naive reset in mammals.

Insights into the Glutamatergic and GABAergic Connections in the Conductance-Based Model of Subthalamic Nucleus in Hyperdirect Pathway.

Introduction: This study aimed to analyze the spiking patterns of subthalamic nucleus and globus pallidus coupling in hyperdirect pathway in healthy primates and in Parkinson's disease using a conductance-based model. The effect of calcium membrane potential has also been investigated. Materials and Methods: System of coupled differential equation arising from the conductance-based model has been simulated using ODE45 in MATLAB 7.14 to analyze the spiking patterns. Results: Analysis of spiking patterns suggests that subthalamic nucleus with synaptic input from globus pallidus in hyperdirect pathways is capable of showing two types of spiking pattern - irregular and rhythmic. Characterization of spiking patterns in healthy and Parkinson condition has been done based on their frequency, trend, and spiking rate. Results indicate that rhythmic patterns does not account for Parkinson's disease. Further, calcium membrane potential is an important parameter to target for identifying the cause of this disease. Conclusion: This work demonstrates that subthalamic nucleus and globus pallidus coupling in hyperdirect pathway can account for Parkinson's symptoms. However, the entire process of excitations and inhibition caused by glutamate and GABA receptors is limited by the timing of depolarization of the model. There is improvement in the correlation between healthy and Parkinson's patterns by increase in calcium membrane potential, however, for a limited time.

Physics of large thermoelectric power factors in SnSe nanoflakes in mid-temperature range.

We have theoretically investigated the underlying physics of observed high electrical conductivity (σ), simultaneous increase of σ and Seebeck coefficient (S) with temperature, and large power factors (PFs) in nominally undoped SnSe nanoflakes sintered at different temperatures, reported recently in Mandavaet al(2022Nanotechnology33155710). Given the fact that S and σ show unusual temperature trends and that the undoped SnSe samples are highly porous and disordered, the conventional Boltzmann theory does not appear to be an appropriate model to describe their transport properties. We have, instead, used a strong disorder model based on percolation theory where charge and energy transport take place through hopping between localized states to understand these observations. Our model is able to explain the observed temperature dependence of σ and S with temperature. Large σ can be explained by a high density of localized states and a large hopping rate. The sample sintered at a higher temperature has lower disorder (σDOS) and higher hopping rate (1/τ0). We findσDOS= 0.151 eV and 1/τ0= 0.143 × 1015s-1for sample sintered at 673 K andσDOS= 0.044 eV and 1/τ0= 2.023 × 1015s-1for sample sintered at 703 K. These values are comparable to the reported values of transition frequencies, confirming that the dominant charge transport mechanism in these SnSe nanoflakes is hopping transport. Finally, we suggest that hopping transport via localized states can result in enhanced thermoelectric properties in disordered polycrystalline materials.

ZFP281 coordinates DNMT3 and TET1 for transcriptional and epigenetic control in pluripotent state transitions.

The progression from naive through formative to primed in vitro pluripotent stem cell states recapitulates the development of the epiblast in vivo during the peri-implantation period of mammalian development. Activation of the de novo DNA methyltransferases and reorganization of transcriptional and epigenetic landscapes are key events occurring during these pluripotent state transitions. However, the upstream regulators that coordinate these events are relatively underexplored. Here, using Zfp281 knockout mouse and degron knock-in cell models, we uncover the direct transcriptional activation of Dnmt3a/3b by ZFP281 in pluripotent stem cells. Chromatin co-occupancy of ZFP281 and DNA hydroxylase TET1, dependent on the formation of R loops in ZFP281-targeted gene promoters, undergoes a "high-low-high" bimodal pattern regulating dynamic DNA methylation and gene expression during the naïive-formative-primed transitions. ZFP281 also safeguards DNA methylation in maintaining primed pluripotency. Our study demonstrates a previously unappreciated role for ZFP281 in coordinating DNMT3A/3B and TET1 functions to promote pluripotent state transitions.

The pluripotency factor Tex10 finetunes Wnt signaling for PGC and male germline development.

Testis-specific transcript 10 (Tex10) is a critical factor for pluripotent stem cell maintenance and preimplantation development. Here, we dissect its late developmental roles in primordial germ cell (PGC) specification and spermatogenesis using cellular and animal models. We discover that Tex10 binds the Wnt negative regulator genes, marked by H3K4me3, at the PGC-like cell (PGCLC) stage in restraining Wnt signaling. Depletion and overexpression of Tex10 hyperactivate and attenuate the Wnt signaling, resulting in compromised and enhanced PGCLC specification efficiency, respectively. Using the Tex10 conditional knockout mouse models combined with single-cell RNA sequencing, we further uncover critical roles of Tex10 in spermatogenesis with Tex10 loss causing reduced sperm number and motility associated with compromised round spermatid formation. Notably, defective spermatogenesis in Tex10 knockout mice correlates with aberrant Wnt signaling upregulation. Therefore, our study establishes Tex10 as a previously unappreciated player in PGC specification and male germline development by fine-tuning Wnt signaling.

Mild sleep restriction increases endothelial oxidative stress in female persons.

Sleep restriction is associated with increased cardiovascular risk, which is more pronounced in female than male persons. We reported recently first causal evidence that mild, prolonged sleep restriction mimicking "real-life" conditions impairs endothelial function, a key step in the development and progression of cardiovascular disease, in healthy female persons. However, the underlying mechanisms are unclear. In model organisms, sleep restriction increases oxidative stress and upregulates antioxidant response via induction of the antioxidant regulator nuclear factor (erythroid-derived 2)-like 2 (Nrf2). Here, we assessed directly endothelial cell oxidative stress and antioxidant responses in healthy female persons (n = 35) after 6 weeks of mild sleep restriction (1.5 h less than habitual sleep) using randomized crossover design. Sleep restriction markedly increased endothelial oxidative stress without upregulating antioxidant response. Using RNA-seq and a predicted protein-protein interaction database, we identified reduced expression of endothelial Defective in Cullin Neddylation-1 Domain Containing 3 (DCUN1D3), a protein that licenses Nrf2 antioxidant responses, as a mediator of impaired endothelial antioxidant response in sleep restriction. Thus, sleep restriction impairs clearance of endothelial oxidative stress that over time increases cardiovascular risk.Trial Registration: NCT02835261 .

15 minute consultation: a structured approach to the management of facial paralysis in a child.

OBJECTIVE: To present a structured approach for an outpatient consultation of a child with facial paralysis. METHOD: Review of literature and description of approach followed in our unit. CONCLUSION: A focused history and examination is key to establish the cause and draw a management plan for paediatric facial paralysis.