Liver Immune Profiling Reveals Pathogenesis and Therapeutics for Biliary Atresia.

Biliary atresia (BA) is a severe cholangiopathy that leads to liver failure in infants, but its pathogenesis remains to be fully characterized. By single-cell RNA profiling, we observed macrophage hypo-inflammation, Kupffer cell scavenger function defects, cytotoxic T cell expansion, and deficiency of CX3CR1+effector T and natural killer (NK) cells in infants with BA. More importantly, we discovered that hepatic B cell lymphopoiesis did not cease after birth and that tolerance defects contributed to immunoglobulin G (IgG)-autoantibody accumulation in BA. In a rhesus-rotavirus induced BA model, depleting B cells or blocking antigen presentation ameliorated liver damage. In a pilot clinical study, we demonstrated that rituximab was effective in depleting hepatic B cells and restoring the functions of macrophages, Kupffer cells, and T cells to levels comparable to those of control subjects. In summary, our comprehensive immune profiling in infants with BA had educed that B-cell-modifying therapies may alleviate liver pathology.

Blockade of the checkpoint receptor TIGIT prevents NK cell exhaustion and elicits potent anti-tumor immunity.

Checkpoint blockade enhances effector T cell function and has elicited long-term remission in a subset of patients with a broad spectrum of cancers. TIGIT is a checkpoint receptor thought to be involved in mediating T cell exhaustion in tumors; however, the relevance of TIGIT to the dysfunction of natural killer (NK) cells remains poorly understood. Here we found that TIGIT, but not the other checkpoint molecules CTLA-4 and PD-1, was associated with NK cell exhaustion in tumor-bearing mice and patients with colon cancer. Blockade of TIGIT prevented NK cell exhaustion and promoted NK cell-dependent tumor immunity in several tumor-bearing mouse models. Furthermore, blockade of TIGIT resulted in potent tumor-specific T cell immunity in an NK cell-dependent manner, enhanced therapy with antibody to the PD-1 ligand PD-L1 and sustained memory immunity in tumor re-challenge models. This work demonstrates that TIGIT constitutes a previously unappreciated checkpoint in NK cells and that targeting TIGIT alone or in combination with other checkpoint receptors is a promising anti-cancer therapeutic strategy.

CRISPR Inversion of CTCF Sites Alters Genome Topology and Enhancer/Promoter Function.

CTCF and the associated cohesin complex play a central role in insulator function and higher-order chromatin organization of mammalian genomes. Recent studies identified a correlation between the orientation of CTCF-binding sites (CBSs) and chromatin loops. To test the functional significance of this observation, we combined CRISPR/Cas9-based genomic-DNA-fragment editing with chromosome-conformation-capture experiments to show that the location and relative orientations of CBSs determine the specificity of long-range chromatin looping in mammalian genomes, using protocadherin (Pcdh) and ß-globin as model genes. Inversion of CBS elements within the Pcdh enhancer reconfigures the topology of chromatin loops between the distal enhancer and target promoters and alters gene-expression patterns. Thus, although enhancers can function in an orientation-independent manner in reporter assays, in the native chromosome context, the orientation of at least some enhancers carrying CBSs can determine both the architecture of topological chromatin domains and enhancer/promoter specificity. These findings reveal how 3D chromosome architecture can be encoded by linear genome sequences.

Systematic functional characterization of antisense eRNA of protocadherin α composite enhancer.

Enhancers generate bidirectional noncoding enhancer RNAs (eRNAs) that may regulate gene expression. At present, the eRNA function remains enigmatic. Here, we report a 5' capped antisense eRNA PEARL (Pcdh eRNA associated with R-loop formation) that is transcribed from the protocadherin (Pcdh) α HS5-1 enhancer region. Through loss- and gain-of-function experiments with CRISPR/Cas9 DNA fragment editing, CRISPRi, and CRISPRa, as well as locked nucleic acid strategies, in conjunction with ChIRP, MeDIP, DRIP, QHR-4C, and HiChIP experiments, we found that PEARL regulates Pcdhα gene expression by forming local RNA-DNA duplexes (R-loops) in situ within the HS5-1 enhancer region to promote long-distance chromatin interactions between distal enhancers and target promoters. In particular, increased levels of eRNA PEARL via perturbing transcription elongation factor SPT6 lead to strengthened local three-dimensional chromatin organization within the Pcdh superTAD. These findings have important implications regarding molecular mechanisms by which the HS5-1 enhancer regulates stochastic Pcdhα promoter choice in single cells in the brain.

Patterned cPCDH expression regulates the fine organization of the neocortex.

The neocortex consists of a vast number of diverse neurons that form distinct layers and intricate circuits at the single-cell resolution to support complex brain functions1. Diverse cell-surface molecules are thought to be key for defining neuronal identity, and they mediate interneuronal interactions for structural and functional organization2-6. However, the precise mechanisms that control the fine neuronal organization of the neocortex remain largely unclear. Here, by integrating in-depth single-cell RNA-sequencing analysis, progenitor lineage labelling and mosaic functional analysis, we report that the diverse yet patterned expression of clustered protocadherins (cPCDHs)-the largest subgroup of the cadherin superfamily of cell-adhesion molecules7-regulates the precise spatial arrangement and synaptic connectivity of excitatory neurons in the mouse neocortex. The expression of cPcdh genes in individual neocortical excitatory neurons is diverse yet exhibits distinct composition patterns linked to their developmental origin and spatial positioning. A reduction in functional cPCDH expression causes a lateral clustering of clonally related excitatory neurons originating from the same neural progenitor and a significant increase in synaptic connectivity. By contrast, overexpression of a single cPCDH isoform leads to a lateral dispersion of clonally related excitatory neurons and a considerable decrease in synaptic connectivity. These results suggest that patterned cPCDH expression biases fine spatial and functional organization of individual neocortical excitatory neurons in the mammalian brain.

DNA Damage Promotes TMPRSS2-ERG Oncoprotein Destruction and Prostate Cancer Suppression via Signaling Converged by GSK3ß and WEE1.

TMPRSS2-ERG gene fusion occurs in approximately 50% of cases of prostate cancer (PCa), and the fusion product is a key driver of prostate oncogenesis. However, how to leverage cellular signaling to ablate TMPRSS2-ERG oncoprotein for PCa treatment remains elusive. Here, we demonstrate that DNA damage induces proteasomal degradation of wild-type ERG and TMPRSS2-ERG oncoprotein through ERG threonine-187 and tyrosine-190 phosphorylation mediated by GSK3ß and WEE1, respectively. The dual phosphorylation triggers ERG recognition and degradation by the E3 ubiquitin ligase FBW7 in a manner independent of a canonical degron. DNA damage-induced TMPRSS2-ERG degradation was abolished by cancer-associated PTEN deletion or GSK3ß inactivation. Blockade of DNA damage-induced TMPRSS2-ERG oncoprotein degradation causes chemotherapy-resistant growth of fusion-positive PCa cells in culture and in mice. Our findings uncover a previously unrecognized TMPRSS2-ERG protein destruction mechanism and demonstrate that intact PTEN and GSK3ß signaling are essential for effective targeting of ERG protein by genotoxic therapeutics in fusion-positive PCa.

Confluence and convergence of Dscam and Pcdh cell-recognition codes.

The ability of neurites of the same neuron to avoid each other (self-avoidance) is a conserved feature in both invertebrates and vertebrates. The key to self-avoidance is the generation of a unique subset of cell-surface proteins in individual neurons engaging in isoform-specific homophilic interactions that drive neurite repulsion rather than adhesion. Among these cell-surface proteins are fly Dscam1 and vertebrate clustered protocadherins (cPcdhs), as well as the recently characterized shortened Dscam (sDscam) in the Chelicerata. Herein, we review recent advances in our understanding of how cPcdh, Dscam, and sDscam cell-surface recognition codes are expressed and translated into cellular functions essential for neural wiring.

Phosphorylated RB Promotes Cancer Immunity by Inhibiting NF-κB Activation and PD-L1 Expression.

Aberrant expression of programmed death ligand-1 (PD-L1) in tumor cells promotes cancer progression by suppressing cancer immunity. The retinoblastoma protein RB is a tumor suppressor known to regulate the cell cycle, DNA damage response, and differentiation. Here, we demonstrate that RB interacts with nuclear factor κB (NF-κB) protein p65 and that their interaction is primarily dependent on CDK4/6-mediated serine-249/threonine-252 (S249/T252) phosphorylation of RB. RNA-seq analysis shows a subset of NF-κB pathway genes including PD-L1 are selectively upregulated by RB knockdown or CDK4/6 inhibitor. S249/T252-phosphorylated RB inversely correlates with PD-L1 expression in patient samples. Expression of a RB-derived S249/T252 phosphorylation-mimetic peptide suppresses radiotherapy-induced upregulation of PD-L1 and augments therapeutic efficacy of radiation in vivo. Our findings reveal a previously unrecognized tumor suppressor function of hyperphosphorylated RB in suppressing NF-κB activity and PD-L1 expression and suggest that the RB-NF-κB axis can be exploited to overcome cancer immune evasion triggered by conventional or targeted therapies.

Precise and Predictable CRISPR Chromosomal Rearrangements Reveal Principles of Cas9-Mediated Nucleotide Insertion.

Chromosomal rearrangements including large DNA-fragment inversions, deletions, and duplications by Cas9 with paired sgRNAs are important to investigate genome structural variations and developmental gene regulation, but little is known about the underlying mechanisms. Here, we report that disrupting CtIP or FANCD2, which have roles in alternative non-homologous end joining, enhances precise DNA-fragment deletion. By analyzing the inserted nucleotides at the junctions of DNA-fragment editing of deletions, inversions, and duplications and characterizing the cleaved products, we find that Cas9 endonucleolytically cleaves the noncomplementary strand with a flexible scissile profile upstream of the -3 position of the PAM site in vivo and in vitro, generating double-strand break ends with 5' overhangs of 1-3 nucleotides. Moreover, we find that engineered Cas9 nucleases have distinct cleavage profiles. Finally, Cas9-mediated nucleotide insertions are nonrandom and are equal to the combined sequences upstream of both PAM sites with predicted frequencies. Thus, precise and predictable DNA-fragment editing could be achieved by perturbing DNA repair genes and using appropriate PAM configurations.

Quantifying the partial ionization effect of gold in the transition region between condensed matter and warm dense matter.

It is now well known that solids under ultra-high-pressure shock compression will enter the warm dense matter (WDM) regime which connects condensed matter and hot plasma. How condensed matter turns into the WDM, however, remains largely unexplored due to the lack of data in the transition pressure range. In this letter, by employing the unique high-Z three-stage gas gun launcher technique developed recently, we compress gold into TPa shock pressure to fill the gap inaccessible by the two-stage gas gun and laser shock experiments. With the aid of high-precision Hugoniot data obtained experimentally, we observe a clear softening behavior beyond ~560 GPa. The state-of-the-art ab-initio molecular dynamics calculations reveal that the softening is caused by the ionization of 5d electrons in gold. This work quantifies the partial ionization effect of electrons under extreme conditions, which is critical to model the transition region between condensed matter and WDM.

Collective motion in hcp-Fe at Earth's inner core conditions.

Earth's inner core is predominantly composed of solid iron (Fe) and displays intriguing properties such as strong shear softening and an ultrahigh Poisson's ratio. Insofar, physical mechanisms to explain these features coherently remain highly debated. Here, we have studied longitudinal and shear wave velocities of hcp-Fe (hexagonal close-packed iron) at relevant pressure-temperature conditions of the inner core using in situ shock experiments and machine learning molecular dynamics (MLMD) simulations. Our results demonstrate that the shear wave velocity of hcp-Fe along the Hugoniot in the premelting condition, defined as T/Tm (Tm: melting temperature of iron) above 0.96, is significantly reduced by ~30%, while Poisson's ratio jumps to approximately 0.44. MLMD simulations at 230 to 330 GPa indicate that collective motion with fast diffusive atomic migration occurs in premelting hcp-Fe primarily along [100] or [010] crystallographic direction, contributing to its elastic softening and enhanced Poisson's ratio. Our study reveals that hcp-Fe atoms can diffusively migrate to neighboring positions, forming open-loop and close-loop clusters in the inner core conditions. Hcp-Fe with collective motion at the inner core conditions is thus not an ideal solid previously believed. The premelting hcp-Fe with collective motion behaves like an extremely soft solid with an ultralow shear modulus and an ultrahigh Poisson's ratio that are consistent with seismic observations of the region. Our findings indicate that premelting hcp-Fe with fast diffusive motion represents the underlying physical mechanism to help explain the unique seismic and geodynamic features of the inner core.

Integrated Bioinformatics and Machine Learning Analysis Identify ACADL as a Potent Biomarker of Reactive Mesothelial Cells.

Mesothelial cells with reactive hyperplasia are difficult to distinguish from malignant mesothelioma cells based on cell morphology. This study aimed to identify and validate potential biomarkers that distinguish mesothelial cells from mesothelioma cells through machine learning combined with immunohistochemistry. It integrated the gene expression matrix from three Gene Expression Omnibus data sets (GSE2549, GSE12345, and GSE51024) to analyze the differently expressed genes between normal and mesothelioma tissues. Then, three machine learning algorithms, least absolute shrinkage and selection operator, support vector machine recursive feature elimination, and random forest were used to screen and obtain four shared candidate markers, including ACADL, EMP2, GPD1L, and HMMR. The receiver operating characteristic curve analysis showed that the area under the curve for distinguishing normal mesothelial cells from mesothelioma was 0.976, 0.943, 0.962, and 0.956, respectively. The expression and diagnostic performance of these candidate genes were validated in two additional independent data sets (GSE42977 and GSE112154), indicating that the performances of ACADL, GPD1L, and HMMR were consistent between the training and validation data sets. Finally, the optimal candidate marker ACADL was verified by immunohistochemistry assay. Acyl-CoA dehydrogenase long chain (ACADL) was stained strongly in mesothelial cells, especially for reactive hyperplasic mesothelial cells, but was negative in malignant mesothelioma cells. Therefore, ACADL has the potential to be used as a specific marker of reactive hyperplasic mesothelial cells in the differential diagnosis of mesothelioma.

Silencing of FUN14 Domain Containing 1 Inhibits Platelet Activation in Diabetes Mellitus through Blocking Mitophagy.

Platelet hyperactivity represents a deleterious physiological phenomenon in diabetes mellitus (DM). This study aimed to explore the role of FUN14 domain containing 1 (FUNDC1) in platelet activation within the context of DM and to uncover relevant mechanisms, with a focus on mitophagy. A mouse model of DM was established by high-fat feeding and streptozotocin injection. Platelets isolated from whole blood were exposed to carbonyl cyanide-4-(trifluo-romethoxy)phenylhydrazone (FCCP) to induce mitophagy. The relative mRNA expression of FUNDC1 was detected by quantitative real-time PCR (qRT-PCR). Western blotting was employed to measure the protein levels of FUNDC1, the ratio of LC3-II toLC3-I, and cleaved caspase-3. Immunofluorescence and flow cytometry were performed to assess LC3-positive mitochondria and platelet activation factor CD62P, respectively. Additionally, serum levels of ß-thrombo-globulin (ß-TG) and platelet factor 4 (PF4)were measured by enzyme-linked immunosorbent assay. FUNDC1 expression was elevated in DM mice, and its silencing decreased the body weight and fasting blood glucose. Inhibition of FUNDC1 also significantly attenuated FCCP-induced platelet mitophagy, as evidenced by the down-regulation of the LC3-II/LC3-I ratio, up-regulation of Tomm20, and diminished presence of LC3-positive mitochondria. Moreover, platelet activation was noted in DM mice; this activation was mitigated upon FUNDC1 silencing, which was confirmed by the down-regulation of cleaved caspase-3 and CD62P as well as reductions in ß-TG and PF4 serum levels. Silencing of FUNDC1 inhibited platelet hyperactivity in DM by impeding mitophagy. As such, FUNDC1-midiated mitophagy may be a promising target for the treatment of DM and its associated cardiovascular complications related cardiovascular events.

BRD9-mediated control of the TGF-ß/Activin/Nodal pathway regulates self-renewal and differentiation of human embryonic stem cells and progression of cancer cells.

Bromodomain-containing protein 9 (BRD9) is a specific subunit of the non-canonical SWI/SNF (ncBAF) chromatin-remodeling complex, whose function in human embryonic stem cells (hESCs) remains unclear. Here, we demonstrate that impaired BRD9 function reduces the self-renewal capacity of hESCs and alters their differentiation potential. Specifically, BRD9 depletion inhibits meso-endoderm differentiation while promoting neural ectoderm differentiation. Notably, supplementation of NODAL, TGF-ß, Activin A or WNT3A rescues the differentiation defects caused by BRD9 loss. Mechanistically, BRD9 forms a complex with BRD4, SMAD2/3, ß-CATENIN and P300, which regulates the expression of pluripotency genes and the activity of TGF-ß/Nodal/Activin and Wnt signaling pathways. This is achieved by regulating the deposition of H3K27ac on associated genes, thus maintaining and directing hESC differentiation. BRD9-mediated regulation of the TGF-ß/Activin/Nodal pathway is also demonstrated in the development of pancreatic and breast cancer cells. In summary, our study highlights the crucial role of BRD9 in the regulation of hESC self-renewal and differentiation, as well as its participation in the progression of pancreatic and breast cancers.

Oxygen-Bridged Cu Binuclear Sites for Efficient Electrocatalytic CO2 Reduction to Ethanol at Ultralow Overpotential.

Electrocatalytic CO2 reduction (CO2RR) to alcohols offers a promising strategy for converting waste CO2 into valuable fuels/chemicals but usually requires large overpotentials. Herein, we report a catalyst comprising unique oxygen-bridged Cu binuclear sites (CuOCu-N4) with a Cu···Cu distance of 3.0-3.1 Å and concomitant conventional Cu-N4 mononuclear sites on hierarchical nitrogen-doped carbon nanocages (hNCNCs). The catalyst exhibits a state-of-the-art low overpotential of 0.19 V (versus reversible hydrogen electrode) for ethanol and an outstanding ethanol Faradaic efficiency of 56.3% at an ultralow potential of -0.30 V, with high-stable Cu active-site structures during the CO2RR as confirmed by operando X-ray adsorption fine structure characterization. Theoretical simulations reveal that CuOCu-N4 binuclear sites greatly enhance the C-C coupling at low potentials, while Cu-N4 mononuclear sites and the hNCNC support increase the local CO concentration and ethanol production on CuOCu-N4. This study provides a convenient approach to advanced Cu binuclear site catalysts for CO2RR to ethanol with a deep understanding of the mechanism.

Solution-Processed 1D Wurtzite ZnS Nanostructures with Controlled Crystallographic Orientation and Tunable Band-Edge Emission.

1D compound semiconductor nanomaterials possess unique physicochemical properties that strongly depend on their size, composition, and structures. ZnS has been widely investigated as one of the most important semiconductors, and the control of crystallographic orientation of 1D ZnS nanostructures is still challenging and crucial to exploring their anisotropic properties. Herein, a solution-processed strategy is developed to synthesize 1D wurtzite (w-)ZnS nanostructures with the specific <002> and <210> orientations by co-decomposing the copper dibutyldithiocarbamate {[(C4 H9 )2 NCS2 ]2 Cu, i.e., R2 Cu} and zinc dibutyldithiocarbamate (R2 Zn) precursors in the mixed solvents of oleylamine and 1-dodecanethoil. A solution-solid-solid (SSS)-Oriented growth mechanism is proposed, which includes oriented nucleation dominated and SSS growth dominated stages. The crystallographic orientation mainly depends on the interfacial energy and ligand effect. The 1D w-ZnS nanostructures with controlled crystallographic orientation display unique morphologies, i.e., <002>-oriented w-ZnS nanorod enclosed with {110} facets while <210>-oriented w-ZnS nanobelt enclosed with wide (002) and narrow (110) facets. The bandgap of 1D w-ZnS nanostructures can be tuned from 3.94 to 3.82 eV with the crystallographic growth direction varied from <002> to <210>, thus leading to the tunable band-edge emission from ≈338 to ≈345 nm.

Constructing Gold Single-Atom Catalysts on Hierarchical Nitrogen-Doped Carbon Nanocages for Carbon Dioxide Electroreduction to Syngas.

Precious-metal single-atom catalysts (SACs), featured by high metal utilization and unique coordination structure for catalysis, demonstrate distinctive performances in the fields of heterogeneous and electrochemical catalysis. Herein, gold SACs are constructed on hierarchical nitrogen-doped carbon nanocages (hNCNC) via a simple impregnation-drying process and first exploited for electrocatalytic carbon dioxide reduction reaction (CO2RR) to produce syngas. The as-constructed Au SAC exhibits the high mass activity of 3319 A g-1 Au at -1.0 V (vs reversible hydrogen electrode, RHE), much superior to the Au nanoparticles supported on hNCNC. The ratio of H2/CO can be conveniently regulated in the range of 0.4-2.2 by changing the applied potential. Theoretical study indicates such a potential-dependent H2/CO ratio is attributed to the different responses of HER and CO2RR on Au single-atom sites coordinating with one N atom at the edges of micropores across the nanocage shells. The catalytic mechanism of the Au active sites is associated with the smooth switch between twofold and fourfold coordination during CO2RR, which much decreases the free energy changes of the rate-determining steps and promotes the reaction activity.

An RRM domain protein SOE suppresses transgene silencing in rice.

Gene expression is regulated at multiple levels, including RNA processing and DNA methylation/demethylation. How these regulations are controlled remains unclear. Here, through analysis of a suppressor for the OsEIN2 over-expressor, we identified an RNA recognition motif protein SUPPRESSOR OF EIN2 (SOE). SOE is localized in nuclear speckles and interacts with several components of the spliceosome. We find SOE associates with hundreds of targets and directly binds to a DNA glycosylase gene DNG701 pre-mRNA for efficient splicing and stabilization, allowing for subsequent DNG701-mediated DNA demethylation of the transgene promoter for proper gene expression. The V81M substitution in the suppressor mutant protein mSOE impaired its protein stability and binding activity to DNG701 pre-mRNA, leading to transgene silencing. SOE mutation enhances grain size and yield. Haplotype analysis in c. 3000 rice accessions reveals that the haplotype 1 (Hap 1) promoter is associated with high 1000-grain weight, and most of the japonica accessions, but not indica ones, have the Hap 1 elite allele. Our study discovers a novel mechanism for the regulation of gene expression and provides an elite allele for the promotion of yield potentials in rice.

Transient cavity-cavity strong coupling at terahertz frequency on LiNbO3 chips.

Terahertz (THz) microcavities have garnered considerable attention for their ability to localize and confine THz waves, allowing for strong coupling to remarkably enhance the light-matter interaction. These properties hold great promise for advancing THz science and technology, particularly for high-speed integrated THz chips where transient interaction between THz waves and matter is critical. However, experimental study of these transient time-domain processes requires high temporal and spatial resolution since these processes, such as THz strong coupling, occur in several picoseconds and microns. Thus, most literature studies rarely cover temporal and spatial processes at the same time. In this work, we thoroughly investigate the transient cavity-cavity strong-coupling phenomena at THz frequency and find a Rabi-like oscillation in the microcavities, manifested by direct observation of a periodic energy exchange process via a phase-contrast time-resolved imaging system. Our explanation, based on the Jaynes-Cummings model, provides theoretical insight into this transient strong-coupling process. This work provides an opportunity to deeply understand the transient strong-coupling process between THz microcavities, which sheds light on the potential of THz microcavities for high-speed THz sensor and THz chip design.

All-optical polarization scrambler based on polarization beam splitting with an amplified fiber ring.

Optical-fiber-based polarization scramblers can reduce the impact of polarization sensitive performance of various optical fiber systems. Here, we propose a simple and efficient polarization scrambler based on an all-optical Mach-Zehnder structure by combining a polarization beam splitter and an amplified fiber ring. To totally decoherence one polarization split beam, a fiber ring together with an amplifier is incorporated. The ratio of two orthogonal beams can be controlled by varying the amplification factor, and we observe different evolution trajectories of the output state of polarizations on the Poincaré sphere. When the amplification factor exceeds a certain threshold, the scrambler system exhibits nearly ideal polarization scrambling behavior. A commercial single wavelength laser with a linewidth of 3 MHz is utilized to characterize the scrambling performance. We found that when the sampling rate is 1.6 MSa/s, a scrambling speed up to 2000krad/s can be obtained for the average degree of polarization being less than 0.1. We also exploit these random polarization fluctuations to generate random binary numbers, indicating that the proposed technique is a good candidate for a random bit generator.