How autoreactive thymocytes differentiate into regulatory versus effector CD4+ T cells after avoiding clonal deletion.

Thymocytes bearing autoreactive T cell receptors (TCRs) are agonist-signaled by TCR/co-stimulatory molecules to either undergo clonal deletion or to differentiate into specialized regulatory T (Treg) or effector T (Teff) CD4+ cells. How these different fates are achieved during development remains poorly understood. We now document that deletion and differentiation are agonist-signaled at different times during thymic selection and that Treg and Teff cells both arise after clonal deletion as alternative lineage fates of agonist-signaled CD4+CD25+ precursors. Disruption of agonist signaling induces CD4+CD25+ precursors to initiate Foxp3 expression and become Treg cells, whereas persistent agonist signaling induces CD4+CD25+ precursors to become IL-2+ Teff cells. Notably, we discovered that transforming growth factor-ß induces Foxp3 expression and promotes Treg cell development by disrupting weaker agonist signals and that Foxp3 expression is not induced by IL-2 except under non-physiological in vivo conditions. Thus, TCR signaling disruption versus persistence is a general mechanism of lineage fate determination in the thymus that directs development of agonist-signaled autoreactive thymocytes.

SLC25A51 is a mammalian mitochondrial NAD+ transporter.

Mitochondria require nicotinamide adenine dinucleotide (NAD+) to carry out the fundamental processes that fuel respiration and mediate cellular energy transduction. Mitochondrial NAD+ transporters have been identified in yeast and plants1,2, but their existence in mammals remains controversial3-5. Here we demonstrate that mammalian mitochondria can take up intact NAD+, and identify SLC25A51 (also known as MCART1)-an essential6,7 mitochondrial protein of previously unknown function-as a mammalian mitochondrial NAD+ transporter. Loss of SLC25A51 decreases mitochondrial-but not whole-cell-NAD+ content, impairs mitochondrial respiration, and blocks the uptake of NAD+ into isolated mitochondria. Conversely, overexpression of SLC25A51 or SLC25A52 (a nearly identical paralogue of SLC25A51) increases mitochondrial NAD+ levels and restores NAD+ uptake into yeast mitochondria lacking endogenous NAD+ transporters. Together, these findings identify SLC25A51 as a mammalian transporter capable of importing NAD+ into mitochondria.

CCAAT Promoter element regulates transgenerational expression of the MHC class I gene.

Transgenerational gene expression depends on both underlying DNA sequences and epigenetic modifications. The latter, which can result in transmission of variegated gene expression patterns across multiple generations without DNA alterations, has been termed epigenetic inheritance and has been documented in plants, worms, flies and mammals. Whereas transcription factors binding to cognate DNA sequence elements regulate gene expression, the molecular basis for epigenetic inheritance has been linked to histone and DNA modifications and non-coding RNA. Here we report that mutation of the CCAAT box promoter element abrogates NF-Y binding and disrupts the stable transgenerational expression of an MHC class I transgene. Transgenic mice with a mutated CCAAT box in the MHC class I transgene display variegated expression of the transgene among littermates and progeny in multiple independently derived transgenic lines. After 4 generations, CCAAT mutant transgenic lines derived from a single founder stably displayed distinct patterns of expression. Histone modifications and RNA polymerase II binding correlate with expression of CCAAT mutant transgenic lines, whereas DNA methylation and nucleosome occupancy do not. Mutation of the CCAAT box also results in changes to CTCF binding and DNA looping patterns across the transgene that correlate with expression status. These studies identify the CCAAT promoter element as a regulator of stable transgenerational gene expression such that mutation of the CCAAT box results in variegated transgenerational inheritance. Considering that the CCAAT box is present in 30% of eukaryotic promoters, this study provides insights into how fidelity of gene expression patterns is maintained through multiple generations.

Screening single-cell trajectories via continuity assessments for cell transition potential.

Advances in single-cell sequencing and data analysis have made it possible to infer biological trajectories spanning heterogeneous cell populations based on transcriptome variation. These trajectories yield a wealth of novel insights into dynamic processes such as development and differentiation. However, trajectory analysis relies on an assumption of trajectory continuity, and experimental limitations preclude some real-world scenarios from meeting this condition. The current lack of assessment metrics makes it difficult to ascertain if/when a given trajectory deviates from continuity, and what impact such a divergence would have on inference accuracy is unclear. By analyzing simulated breaks introduced into in silico and real single-cell data, we found that discontinuity caused precipitous drops in the accuracy of trajectory inference. We then generate a simple scoring algorithm for assessing trajectory continuity, and found that continuity assessments in real-world cases of intestinal stem cell development and CD8 + T cells differentiation efficiently identifies trajectories consistent with empirical knowledge. This assessment approach can also be used in cases where a priori knowledge is lacking to screen a pool of inferred lineages for their adherence to presumed continuity, and serve as a means for weighing higher likelihood trajectories for validation via empirical studies, as exemplified by our case studies in psoriatic arthritis and acute kidney injury. This tool is freely available through github at qingshanni/scEGRET.

Dynamics of SLC25A51 reveal preference for oxidized NAD+ and substrate led transport.

SLC25A51 is a member of the mitochondrial carrier family (MCF) but lacks key residues that contribute to the mechanism of other nucleotide MCF transporters. Thus, how SLC25A51 transports NAD+ across the inner mitochondrial membrane remains unclear. To elucidate its mechanism, we use Molecular Dynamics simulations to reconstitute SLC25A51 homology models into lipid bilayers and to generate hypotheses to test. We observe spontaneous binding of cardiolipin phospholipids to three distinct sites on the exterior of SLC25A51's central pore and find that mutation of these sites impairs cardiolipin binding and transporter activity. We also observe that stable formation of the required matrix gate is controlled by a single salt bridge. We identify binding sites in SLC25A51 for NAD+ and show that its selectivity for NAD+ is guided by an electrostatic interaction between the charged nicotinamide ring in the ligand and a negatively charged patch in the pore. In turn, interaction of NAD+ with interior residue E132 guides the ligand to dynamically engage and weaken the salt bridge gate, representing a ligand-induced initiation of transport.

Multifunctional Chiral Chemically-Powered Micropropellers for Cargo Transport and Manipulation.

Practical applications of synthetic self-propelled nano and microparticles for microrobotics, targeted drug delivery, and manipulation at the nanoscale are rapidly expanding. However, fabrication limitations often hinder progress, resulting in relatively simple shapes and limited functionality. Here, taking advantage of 3D nanoscale printing, chiral micropropellers powered by the hydrogen peroxide reduction reaction are fabricated. Due to their chirality, the propellers exhibit multifunctional behavior controlled by an applied magnetic field: spinning in place (loitering), directed migration in the prescribed direction, capture, and transport of polymer cargo particles. Design parameters of the propellers are optimized by computation modeling based on mesoscale molecular dynamics. It is predicted by computer simulations, and confirmed experimentally, that clockwise rotating propellers attract each other and counterclockwise repel. These results shed light on how chirality and shape optimization enhance the functionality of synthetic autonomous micromachines.

Extracellular Matrix Stiffness Regulates Microvascular Stability by Controlling Endothelial Paracrine Signaling to Determine Pericyte Fate.

BACKGROUND: The differentiation of pericytes into myofibroblasts causes microvascular degeneration, ECM (extracellular matrix) accumulation, and tissue stiffening, characteristics of fibrotic diseases. It is unclear how pericyte-myofibroblast differentiation is regulated in the microvascular environment. Our previous study established a novel 2-dimensional platform for coculturing microvascular endothelial cells (ECs) and pericytes derived from the same tissue. This study investigated how ECM stiffness regulated microvascular ECs, pericytes, and their interactions. METHODS: Primary microvessels were cultured in the TGM2D medium (tubular microvascular growth medium on 2-dimensional substrates). Stiff ECM was prepared by incubating ECM solution in regular culture dishes for 1 hour followed by PBS wash. Soft ECM with Young modulus of ≈6 kPa was used unless otherwise noted. Bone grafts were prepared from the rat skull. Immunostaining, RNA sequencing, RT-qPCR (real-time quantitative polymerase chain reaction), Western blotting, and knockdown experiments were performed on the cells. RESULTS: Primary microvascular pericytes differentiated into myofibroblasts (NG2+αSMA+) on stiff ECM, even with the TGFß (transforming growth factor beta) signaling inhibitor A83-01. Soft ECM and A83-01 cooperatively maintained microvascular stability while inhibiting pericyte-myofibroblast differentiation (NG2+αSMA-/low). We thus defined 2 pericyte subpopulations: primary (NG2+αSMA-/low) and activated (NG2+αSMA+) pericytes. Soft ECM promoted microvascular regeneration and inhibited fibrosis in bone graft transplantation in vivo. As integrins are the major mechanosensor, we performed RT-qPCR screening of integrin family members and found Itgb1 (integrin ß1) was the major subunit downregulated by soft ECM and A83-01 treatment. Knocking down Itgb1 suppressed myofibroblast differentiation on stiff ECM. Interestingly, ITGB1 phosphorylation (Y783) was mainly located on microvascular ECs on stiff ECM, which promoted EC secretion of paracrine factors, including CTGF (connective tissue growth factor), to induce pericyte-myofibroblast differentiation. CTGF knockdown or monoclonal antibody treatment partially reduced myofibroblast differentiation, implying the participation of multiple pathways in fibrosis formation. CONCLUSIONS: ECM stiffness and TGFß signaling cooperatively regulate microvascular stability and pericyte-myofibroblast differentiation. Stiff ECM promotes EC ITGB1 phosphorylation (Y783) and CTGF secretion, which induces pericyte-myofibroblast differentiation.

Templating Influence of Regulated Inorganic Framework in Two-Dimensional Ferroelastic Perovskites: (C3 H5 CH2 NH3 )2 [MCl4 ] (M=Mn and Cd).

The remarkable material stability and structural diversity of two-dimensional (2D) organic-inorganic hybrid perovskites (OIHPs) constitute a vast available library of versatile materials. In particular, ferroelastic property, for which the spontaneous strain can be transformed by applying mechanical stress, is very promising for extensive nanotechnological applications. However, integrating ferroelastic property into 2D OIHPs is still in its infancy. Herein, we designed two new 2D OIHPs (C3 H5 CH2 NH3 )2 [MCl4 ] (M=Mn for 1 and Cd for 2), which undergo reversible ferroelastic phase transitions with an Aizu expression 4/mmmFmmm. The templating influence of the more distorted inorganic framework on the disordering of organic cations and the stronger hydrogen bonds has a key role in the striking improvement of Curie temperature from 246 K in 1 to 273 K in 2. Meanwhile, the minimized alteration of structural motif ensures the well maintaining of the ferroelastic performance in the forms of crystals and thin films, as demonstrated by the identifiable evolution of domain structures. This work will provide a fertile new ground for enlarging the limited number of 2D ferroelastic OIHPs with better practical utility.

Influence of Pd(II) Adsorption on High-Temperature Ferroelastic Phase Transition in (2-Amino-2-thiazolinium)PbBr3.

Ferroelastic materials have received special attention because of their great promise for mechanical switches, piezoelectric sensors, and data storage applications. Here, we report a novel ferroelastic semiconducting hybrid organic-inorganic perovskite (C3H7N2S)PbBr3 (1) [(C3H7N2S)+ is 2-amino-2-thiazolinium] with a ferroelastic phase transition at 395 K and an optical band gap of 3.43 eV. 1 has a one-dimensional BaNiO3-type structure and undergoes a high-temperature ferroelastic phase transition with an Aizu notation of mmmF2/m. Meanwhile, 1 exhibits dielectric switch near the phase transition temperature. By introducing the thioether group, the motion of the molecules or ions of 1 is hindered after the sorption of Pd(II) metal ions, which leads to the disappearance of the high-temperature ferroelastic phase transition and dielectric switch. This is the first reported ferroelastic semiconductor material with Pd(II) adsorption property, by studying the influence of Pd(II) adsorption on high-temperature ferroelastic phase transition, it may be enlightening to further uncover the mechanism of phase transition or the origin of ferroelasticity, which represents an important step toward multifunctional applications of lead-hybrid perovskite-based ferroelastic materials.

Licochalcone A Derivatives as Selective Dipeptidyl Peptidase 4 Inhibitors with Anti-Inflammatory Effects.

A set of 22 analogs of licochalcone A was designed and synthesized to explore their potentials as dipeptidyl peptidase 4 (DPP4) inhibitors with anti-inflammatory effects. The anti-DPP4 effects of these analogs were evaluated using the fluorescent substrate Gly-Pro-N-butyl-4-amino-1,8-naphthalimide (GP-BAN). The nitro-substituted analogue 27 exhibited the most potent activity (Ki = 0.96 µM). A structure-activity relationship investigation revealed that 4-hydroxyl and 5-chloro substituents are essential for DPP4 inhibition, while the 3'-nitro substituent improved both DPP4 inhibition and microsomal stability. Furthermore, compound 27 demonstrated good selectivity for DPP4 over other proteases, including dipeptidyl peptidase 9 (DPP9), thrombin, prolyl endopeptidase (PREP), and fibroblast activation protein (FAP). The cytotoxic effect of 27 was evaluated in cancer cell lines HepG-2 and Caco-2 and in somatic RAW264.7 cells and RPTECs. Compound 27 showed no toxicity to normal cells and weak toxicity to cancer cells. In a living cell imaging assay, 27 blocked the dipeptidase activity of DPP4 in both Caco-2 and HepG-2 cells. This compound also dose-dependently suppressed the expression levels of the chemokines tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1ß (IL-1ß).

Influence of optical surface distortion in a cylindrical Offner stretcher on the far-field signal-to-noise ratio.

In a stretcher, the surface distortion of the optical elements can introduce spectral phase modulations into the laser, which can affect the laser's signal-to-noise ratio. In this paper, by combining ray tracing methods and angular spectrum diffraction methods, the impact of the mid-frequency surface distortion of the optical elements in an cylindrical Offner stretcher on the far-field signal-to-noise ratio of the laser is simulated. The results show that reducing the spatial chirp on the convex cylindrical mirror can effectively improve the far-field signal-to-noise ratio of the laser, and two methods to improve the far-field signal-to-noise ratio are presented.

MYC protein stability is negatively regulated by BRD4.

The protooncogene MYC regulates a variety of cellular processes, including proliferation and metabolism. Maintaining MYC at homeostatic levels is critical to normal cell function; overexpression drives many cancers. MYC stability is regulated through phosphorylation: phosphorylation at Thr58 signals degradation while Ser62 phosphorylation leads to its stabilization and functional activation. The bromodomain protein 4 (BRD4) is a transcriptional and epigenetic regulator with intrinsic kinase and histone acetyltransferase (HAT) activities that activates transcription of key protooncogenes, including MYC We report that BRD4 phosphorylates MYC at Thr58, leading to MYC ubiquitination and degradation, thereby regulating MYC target genes. Importantly, BRD4 degradation, but not inhibition, results in increased levels of MYC protein. Conversely, MYC inhibits BRD4's HAT activity, suggesting that MYC regulates its own transcription by limiting BRD4-mediated chromatin remodeling of its locus. The MYC stabilizing kinase, ERK1, regulates MYC levels directly and indirectly by inhibiting BRD4 kinase activity. These findings demonstrate that BRD4 negatively regulates MYC levels, which is counteracted by ERK1 activation.

Persistent Lipid Accumulation Leads to Persistent Exacerbation of Endoplasmic Reticulum Stress and Inflammation in Progressive NASH via the IRE1α/TRAF2 Complex.

Non-alcoholic steatohepatitis (NASH) is a metabolic disorder that often leads to other severe liver diseases, yet treatment options are limited. Endoplasmic reticulum (ER) stress is an important pathogenetic mechanism of NASH and plays a key role in tandem steatosis as well as liver inflammation. This study aims to develop a progressive NASH model through sustained lipid accumulation and to elucidate its molecular mechanism through IRE1α/TRAF2 complex. Male SD rats were fed a high-fat diet (HFD) for 4, 8, and 12 weeks to induce progressive NASH. MRNA sequencing and PPI analysis were used to screen core genes. Transmission electron microscopy, immunofluorescence staining, ELISA, qRT-PCR, and Western blotting were used at each time point to compare differences between each index of progressive NASH at 4, 8, and 12 weeks. Sustained lipid accumulation led to structural disruption of the ER, a reduction in ER number, and an increase of lipid droplet aggregation in hepatocytes. Persistent lipid accumulation led to a persistent increase in mRNA and protein expression of the IRE1α/TRAF2 complex, IKK/IκB/NF-κB signaling pathway and ASK1/JNK1 signaling pathway, and TNF-α, IL-1ß, and IL-6 also continued to increase. Persistent lipid accumulation led to a persistent exacerbation of ER stress and inflammation in progressive NASH via the IRE1α/TRAF2 complex.

Graph Clustering With Graph Capsule Network.

Graph clustering, which aims to partition a set of graphs into groups with similar structures, is a fundamental task in data analysis. With the great advances made by deep learning, deep graph clustering methods have achieved success. However, these methods have two limitations: (1) they learn graph embeddings by a neural language model that fails to effectively express graph properties, and (2) they treat embedding learning and clustering as two isolated processes, so the learned embeddings are unsuitable for the subsequent clustering. To overcome these limitations, we propose a novel capsule-based graph clustering (CGC) algorithm to cluster graphs. First, we construct a graph clustering capsule network (GCCN) that introduces capsules to capture graph properties. Second, we design an iterative optimization strategy to alternately update the GCCN parameters and clustering assignment parameters. This strategy leads GCCN to learn cluster-oriented graph embeddings. Experimental results show that our algorithm achieves performance superior to that of existing graph clustering algorithms in terms of three standard evaluation metrics: ACC, NMI, and ARI. Moreover, we use visualization results to analyze the effectiveness of the capsules and demonstrate that GCCN can learn cluster-oriented embeddings.

Foxp3 transcription factor is proapoptotic and lethal to developing regulatory T cells unless counterbalanced by cytokine survival signals.

Immune tolerance requires regulatory T (Treg) cells to prevent autoimmune disease, with the transcription factor Foxp3 functioning as the critical regulator of Treg cell development and function. We report here that Foxp3 was lethal to developing Treg cells in the thymus because it induced a unique proapoptotic protein signature (Puma⁺⁺⁺p-Bim⁺⁺p-JNK⁺⁺DUSP6⁻) and repressed expression of prosurvival Bcl-2 molecules. However, Foxp3 lethality was prevented by common gamma chain (γc)-dependent cytokine signals that were present in the thymus in limiting amounts sufficient to support only ∼1 million Treg cells. Consequently, most newly arising Treg cells in the thymus were deprived of this signal and underwent Foxp3-induced death, with Foxp3⁺CD25⁻ Treg precursor cells being the most susceptible. Thus, we identify Foxp3 as a proapoptotic protein that requires developing Treg cells to compete with one another for limiting amounts of γc-dependent survival signals in the thymus.

Association of consciousness impairment and mortality in people with COVID-19.

BACKGROUND: To investigate the association between impairment of consciousness and risk of death in people with COVID-19. METHODS: In this multicentre retrospective study, we enrolled people with confirmed COVID-19 from 44 hospitals in Wuhan and Sichuan, China, between 18 January and 30 March 2020. We extracted demographics, clinical, laboratory data and consciousness level (as measured by the Glasgow Coma Scale (GCS) score) from medical records. We used Cox proportional hazards regression, structural equation modelling and survival time analysis to compare people with different progressions of impaired consciousness. RESULTS: We enrolled 1,143 people (average age 51.3 ± standard deviation 17.1-year-old; 50.3% males), of whom 76 died. Increased mortality risk was identified in people with GCS score between 9 and 14 (hazard ratio (HR) 46.76, p < .001) and below 9 (HR 65.86, p < .001). Pathway analysis suggested a significant direct association between consciousness level and death. Other factors, including age, oxygen saturation level and pH, had indirect associations with death mediated by GCS scores. People who developed impaired consciousness more rapidly either from symptoms onset (<10 days vs. 10-19 days, p = .025, <10 days vs. ≥20 days and 10-19 days vs. ≥20 days, <.001) or deterioration of oxygen saturation (≤2 days vs.>2 days, p = .028) had shorter survival times. CONCLUSION: Altered consciousness and its progression had a direct link with death in COVID-19. Interactions with age, oxygen saturation level and pH suggest possible pathophysiology. Further work to confirm these findings explore prevention strategies and interventions to decrease mortality is warranted.

The potential neurological effect of the COVID-19 vaccines: A review.

The coronavirus disease 2019 caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a pandemic with people infected in almost all countries. The most efficient solution to end this pandemic is a safe and efficient vaccine. Classic platforms are used to develop vaccines including live-attenuated vaccine, inactivated vaccine, protein subunit vaccine, and viral vector. Nucleic acid vaccine uses next-generation platforms for their development. Vaccines are now rushing to the market. Eleven candidates are in advance development. These comprise inactivated vaccines, viral vector vaccine, nucleic acid vaccine, and the protein subunit vaccine platform, which are now quite advanced in trials in various geographic and ethnic populations. The reported severe adverse effects raised the worries about their safety. It becomes critical to know whether these vaccines will cause neurologic disorders like previously recognized vaccine-related demyelinating diseases, fever-induced seizure, and other possible deficits. We reviewed the most promising COVID-2 vaccines with a particular interest in mechanism(s) and adverse effect(s). We exemplify potential neurological problems these vaccines could cause by looking at previous studies. The current evidence indicated a minor risk of the acute neurological disorders after the application. The observation of the long-time effect is still needed.

A Positive Regulatory Feedback Loop between EKLF/KLF1 and TAL1/SCL Sustaining the Erythropoiesis.

The erythroid Krüppel-like factor EKLF/KLF1 is a hematopoietic transcription factor binding to the CACCC DNA motif and participating in the regulation of erythroid differentiation. With combined use of microarray-based gene expression profiling and the promoter-based ChIP-chip assay of E14.5 fetal liver cells from wild type (WT) and EKLF-knockout (Eklf-/-) mouse embryos, we identified the pathways and direct target genes activated or repressed by EKLF. This genome-wide study together with the molecular/cellular analysis of the mouse erythroleukemic cells (MEL) indicate that among the downstream direct target genes of EKLF is Tal1/Scl. Tal1/Scl encodes another DNA-binding hematopoietic transcription factor TAL1/SCL, known to be an Eklf activator and essential for definitive erythroid differentiation. Further identification of the authentic Tal gene promoter in combination with the in vivo genomic footprinting approach and DNA reporter assay demonstrate that EKLF activates the Tal gene through binding to a specific CACCC motif located in its promoter. These data establish the existence of a previously unknow positive regulatory feedback loop between two DNA-binding hematopoietic transcription factors, which sustains mammalian erythropoiesis.

New onset acute symptomatic seizure and risk factors in coronavirus disease 2019: A retrospective multicenter study.

Our aim was to clarify the incidence and risk of acute symptomatic seizures in people with coronavirus disease 2019 (COVID-19). This multicenter retrospective study enrolled people with COVID-19 from January 18 to February 18, 2020 at 42 government-designated hospitals in Hubei province, the epicenter of the epidemic in China; Sichuan province; and Chongqing municipality. Data were collected from medical records by 11 neurologists using a standard case report form. A total of 304 people were enrolled, of whom 108 had a severe condition. None in this cohort had a known history of epilepsy. Neither acute symptomatic seizures nor status epilepticus was observed. Two people had seizurelike symptoms during hospitalization due to acute stress reaction and hypocalcemia, and 84 (27%) had brain insults or metabolic imbalances during the disease course known to increase the risk of seizures. There was no evidence suggesting an additional risk of acute symptomatic seizures in people with COVID-19. Neither the virus nor potential risk factors for seizures seem to be significant risks for the occurrence of acute symptomatic seizures in COVID-19.

Active rotational dynamics of a self-diffusiophoretic colloidal motor.

The dynamics of a spherical chemically-powered synthetic colloidal motor that operates by a self-diffusiophoretic mechanism and has a catalytic domain of arbitrary shape is studied using both continuum theory and particle-based simulations. The motor executes active rotational motion when self-generated concentration gradients and interactions between the chemical species and colloidal motor surface break spherical symmetry. Local variations of chemical reaction rates on the motor catalytic surface with catalytic domain sizes and shapes provide such broken symmetry conditions. A continuum theoretical description of the active rotational motion is given, along with the results of particle-based simulations of the active dynamics. From these results a detailed description of the factors responsible for the active rotational dynamics can be given. Since active rotational motion often plays a significant part in the nature of the collective dynamics of many-motor systems and can be used to control motor motion in targeted cargo transport, our results should find applications beyond those considered here.