Local atomic stacking and symmetry in twisted graphene trilayers.

Moiré superlattices formed by twisting trilayers of graphene are a useful model for studying correlated electron behaviour and offer several advantages over their formative bilayer analogues, including a more diverse collection of correlated phases and more robust superconductivity. Spontaneous structural relaxation alters the behaviour of moiré superlattices considerably and has been suggested to play an important role in the relative stability of superconductivity in trilayers. Here we use an interferometric four-dimensional scanning transmission electron microscopy approach to directly probe the local graphene layer alignment over a wide range of trilayer graphene structures. Our results inform a thorough understanding of how reconstruction modulates the local lattice symmetries crucial for establishing correlated phases in twisted graphene trilayers, evincing a relaxed structure that is markedly different from that proposed previously.

E3 ligase DECREASED GRAIN SIZE 1 promotes degradation of a G-protein subunit and positively regulates grain size in rice.

Grain size is one of the most important traits determining crop yield. However, the mechanism controlling grain size remains unclear. Here, we confirmed the E3 ligase activity of DECREASED GRAIN SIZE 1 (DGS1) in positive regulation of grain size in rice (Oryza sativa) suggested in a previous study. Rice G-protein subunit gamma 2 (RGG2), which negatively regulates grain size, was identified as an interacting protein of DGS1. Biochemical analysis suggested that DGS1 specifically interacts with canonical Gγ subunits (rice G-protein subunit gamma 1 [RGG1] and rice G-protein subunit gamma 2 [RGG2]) rather than non-canonical Gγ subunits (DENSE AND ERECT PANICLE 1 [DEP1], rice G-protein gamma subunit type C 2 [GCC2], GRAIN SIZE 3 [GS3]). We also identified the necessary domains for interaction between DGS1 and RGG2. As an E3 ligase, DGS1 ubiquitinated and degraded RGG2 via a proteasome pathway in several experiments. DGS1 also ubiquitinated RGG2 by its K140, K145 and S147 residues. Thus, this work identified a substrate of the E3 ligase DGS1 and elucidated the post transcriptional regulatory mechanism of the G-protein signalling pathway in the control of grain size.

Targeted next-generation sequencing and long-read HiFi sequencing provide novel insights into clinically significant KLF1 variants.

BACKGROUND: Krüppel-like factor 1 (KLF1), a crucial erythroid transcription factor, plays a significant role in various erythroid changes and haemolytic diseases. The rare erythrocyte Lutheran inhibitor (In(Lu)) blood group phenotype serves as an effective model for identifying KLF1 hypomorphic and loss-of-function variants. In this study, we aimed to analyse the genetic background of the In(Lu) phenotype in a population-based sample group by high-throughput technologies to find potentially clinically significant KLF1 variants. RESULTS: We included 62 samples with In(Lu) phenotype, screened from over 300,000 Chinese blood donors. Among them, 36 samples were sequenced using targeted Next Generation Sequencing (NGS), whereas 19 samples were sequenced using High Fidelity (HiFi) technology. In addition, seven samples were simply sequenced using Sanger sequencing. A total of 29 hypomorphic or loss-of-function variants of KLF1 were identified, 21 of which were newly discovered. All new variants discovered by targeted NGS or HiFi sequencing were validated through Sanger sequencing, and the obtained results were found to be consistent. The KLF1 haplotypes of all new variants were further confirmed using clone sequencing or HiFi sequencing. The lack of functional KLF1 variants detected in the four samples indicates the presence of additional regulatory mechanisms. In addition, some samples exhibited BCAM polymorphisms, which encodes antigens of the Lutheran (LU) blood group system. However, no BCAM mutations which leads to the absence of LU proteins were detected. CONCLUSIONS: High-throughput sequencing methods, particularly HiFi sequencing, were introduced for the first time into genetic analysis of the In(Lu) phenotype. Targeted NGS and HiFi sequencing demonstrated the accuracy of the results, providing additional advantages such as simultaneous analysis of other blood group genes and clarification of haplotypes. Using the In(Lu) phenotype, a powerful model for identifying hypomorphic or loss-of-function KLF1 variants, numerous novel variants have been detected, which have contributed to the comprehensive understanding of KLF1. These clinically significant KLF1 mutations can serve as a valuable reference for the diagnosis of related blood cell diseases.

Twisto-Electrochemical Activity Volcanoes in Trilayer Graphene.

In this work, we develop a twist-dependent electrochemical activity map, combining a low-energy continuum electronic structure model with modified Marcus-Hush-Chidsey kinetics in trilayer graphene. We identify a counterintuitive rate enhancement region spanning the magic angle curve and incommensurate twists in the system geometry. We find a broad activity peak with a ruthenium hexamine redox couple in regions corresponding to both magic angles and incommensurate angles, a result qualitatively distinct from the twisted bilayer case. Flat bands and incommensurability offer new avenues for reaction rate enhancements in electrochemical transformations.

Fully genotyping and screening of clinically important blood-group antigens by MALDI TOF mass spectrometry.

Several molecular biology methods are available for high-throughput blood typing. In this study, we aimed to build a high-throughput blood-group genetic screening system for high-frequency blood-group antigen-negative rare-blood groups in donors and patients. The amplification primers for all blood-type gene fragments involving the selected alleles were designed for detection. Single-base extend primers were also designed based on specific loci. DNA fragments were detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) for the last nucleotide identification of amplification products in the extend step. The accuracy was verified by known samples. Thirty-six random samples were detected by serological tests and sequencing to verify the system stability. After verification, according to the collected known rare-blood-type samples, all the alleles designed to be detected matched with the validated single-nucleotide polymorphisms. The verification tests showed that all genotyping results of the random samples were in accordance with the findings of serotyping and sequencing. Then, 1258 random donor samples were screened by the built typing system after the verification. Three Fy(a-) and four s- were screened out in 1258 random blood samples. The multiple polymerase chain reaction-based MS detection system can be used in rare-blood-type screening with good accuracy and stability.

MR Susceptibility Separation for Quantifying Lesion Paramagnetic and Diamagnetic Evolution in Relapsing-Remitting Multiple Sclerosis.

BACKGROUND: Multiple sclerosis (MS) lesion evolution may involve changes in diamagnetic myelin and paramagnetic iron. Conventional quantitative susceptibility mapping (QSM) can provide net susceptibility distribution, but not the discrete paramagnetic and diamagnetic components. PURPOSE: To apply susceptibility separation (χ separation) to follow lesion evolution in MS with comparison to R2 */R2 ' /QSM. STUDY TYPE: Longitudinal, prospective. SUBJECTS: Twenty relapsing-remitting MS subjects (mean age: 42.5 ± 9.4 years, 13 females; mean years of symptoms: 4.3 ± 1.4 years). FIELD STRENGTH/SEQUENCE: Three-dimensional multiple echo gradient echo (QSM and R2 * mapping), two-dimensional dual echo fast spin echo (R2 mapping), T2 -weighted fluid attenuated inversion recovery, and T1-weighted magnetization prepared gradient echo sequences at 3 T. ASSESSMENT: Data were analyzed from two scans separated by a mean interval of 14.4 ± 2.0 months. White matter lesions on fluid-attenuated inversion recovery were defined by an automatic pipeline, then manually refined (by ZZ/AHW, 3/25 years' experience in MRI), and verified by a radiologist (MN, 25 years' experience in MS). Susceptibility separation yielded the paramagnetic and diamagnetic susceptibility content of each voxel. Lesions were classified into four groups based on the variation of QSM/R2 * or separated into positive/negative components from χ separation. STATISTICAL TESTS: Two-sample paired t tests for assessment of longitudinal differences. Spearman correlation coefficients to assess associations between χ separation and R2 */R2 ' /QSM. Significant level: P < 0.005. RESULTS: A total of 183 lesions were quantified. Categorizing lesions into groups based on χ separation demonstrated significant annual changes in QSM//R2 */R2 ' . When lesions were grouped based on changes in QSM and R2 *, both changing in unison yielded a significant dominant paramagnetic variation and both opposing yielded a dominant diamagnetic variation. Significant Spearman correlation coefficients were found between susceptibility-sensitive MRI indices and χ separation. DATA CONCLUSION: Susceptibility separation changes in MS lesions may distinguish and quantify paramagnetic and diamagnetic evolution, potentially providing additional insight compared to R2 * and QSM alone. LEVEL OF EVIDENCE: 2 TECHNICAL EFFICACY: Stage 2.

Regulation of cervical cancer via G15-mediated inhibition of G protein-coupled estrogen receptor.

Cervical cancer is among the most common gynecological malignancies. G protein-coupled estrogen receptor (GPER) is involved in the development of various tumors; however, its role in cervical cancer remains unclear. We investigated whether G15, an inhibitor of GPER, can regulate its expression and affect cervical cancer progression. We examined the biological behaviors of G15-treated SiHa and HeLa cells using Cell Counting Kit-8, monoclonal proliferation, plate scratching, and Transwell invasion experiments. Western blotting was used to detect the expression of GPER, E-cadherin, N-cadherin, vimentin, Bcl-2, Bax, phosphatidylinositol-3-kinase (PI3K)/AKT, and programmed death ligand 1 (PD-L1). The expression of GPER, E-cadherin, vimentin, and PD-L1 in cervical cancer and adjacent tissues was detected using immunohistochemistry. The correlation between GPER expression and clinicopathological characteristics was analyzed. The expression of GPER in cervical cancer tissues was significantly higher than that in paracancerous tissues, and it was detected in the membrane and cytoplasm of SiHa and HeLa cells. The proliferation, migration, and invasion abilities of SiHa and HeLa cells were reduced after G15 treatment. The G15-treated groups exhibited higher expression of E-cadherin and Bax and lower expression of N-cadherin, vimentin, Bcl-2, GPER, p-PI3K, p-AKT, and PD-L1 than the control group. The expression of E-cadherin was lower and that of vimentin was higher in cancer tissues than in paracancerous tissues; PD-L1 was highly expressed in tumor and stromal cells in cancer tissues but not in paracancerous tissues. G15 functions by regulating the GPER/PI3K/AKT/PD-L1 signaling pathway and may serve as a new immunotherapy for treating patients with cervical cancer.

A novel homozygous splice-site mutation of JK gene leads to Jk(a-b-) phenotype.

OBJECTIVES: This study aimed to investigate the molecular mechanism of the Jk(a-b-) phenotype in a Chinese transfusion patient. BACKGROUND: Many different mutation types relating to Jk(a-b-) phenotype have been reported. However, the splice-site mutation is relatively rare and the related functional verification is lacking. MATERIALS AND METHODS: In this study, the blood sample was collected from a transfusion patient with the Jk(a-b-) phenotype. Serotyping was performed using routine serological methods. The exons sequences and coding regions of the JK gene were amplified using polymerase chain reaction and directly sequenced. To perform a minigene splicing assay, the intronic mutation sequences were cloned into a pSPL3 splice reporting vector. The splicing reporter minigene assay was performed in HEK 293T cells. RESULTS: The Jk(a-b-) phenotype of the blood sample was identified through serological testing. Sequencing results revealed that the sample had a novel homozygous splice-site mutation JK*02N (NM_015865.7: c.663+3A>C). Further analysis, including cDNA sequencing and minigene splicing assay, confirmed that the novel splice-site mutation resulted in exon skipping. Interestingly, different numbers of exons being skipped were obtained by the two methods. CONCLUSION: This study revealed a novel homozygous splicing-site mutation associated with the Jk(a-b-) phenotype in Chinese population. Our results emphasise the importance of the in vitro functional method minigene splicing assay, while also acknowledging its potential limitations when compared to cDNA sequencing.

Energy-Efficient and Highly Reliable Geographic Routing Based on Link Detection and Node Collaborative Scheduling in WSN.

Energy efficiency and data reliability are important indicators to measure network performance in wireless sensor networks. In existing research schemes of routing protocols, the impact of node coverage on the network is often ignored, and the possibility that multiple sensor nodes may sense the same spatial point is not taken into account, which results in a waste of network resources, especially in large-scale networks. Apart from that, the blindness of geographic routing in data transmission has been troubling researchers, which means that the nodes are unable to determine the validity of data transmission. In order to solve the above problems, this paper innovatively combines the routing protocol with the coverage control technique and proposes the node collaborative scheduling algorithm, which fully considers the correlation characteristics between sensor nodes to reduce the number of active working nodes and the number of packets generated, to further reduce energy consumption and network delay and improve packet delivery rate. In order to solve the problem of unreliability of geographic routing, a highly reliable link detection and repair scheme is proposed to check the communication link status and repair the invalid link, which can greatly improve the packet delivery rate and throughput of the network, and has good robustness. A large number of experiments demonstrate the effectiveness and superiority of our proposed scheme and algorithm.

Domain-Dependent Surface Adhesion in Twisted Few-Layer Graphene: Platform for Moiré-Assisted Chemistry.

Twisted van der Waals multilayers are widely regarded as a rich platform to access novel electronic phases thanks to the multiple degrees of freedom available for controlling their electronic and chemical properties. Here, we propose that the stacking domains that form naturally due to the relative twist between successive layers act as an additional "knob" for controlling the behavior of these systems and report the emergence and engineering of stacking domain-dependent surface chemistry in twisted few-layer graphene. Using mid-infrared near-field optical microscopy and atomic force microscopy, we observe a selective adhesion of metallic nanoparticles and liquid water at the domains with rhombohedral stacking configurations of minimally twisted double bi- and trilayer graphene. Furthermore, we demonstrate that the manipulation of nanoparticles located at certain stacking domains can locally reconfigure the moiré superlattice in their vicinity at the micrometer scale. Our findings establish a new approach to controlling moiré-assisted chemistry and nanoengineering.

Hemolytic disease of the newborn due to anti-Jra from a Chinese mother with one novel and one classic heterozygous mutation.

OBJECTIVE: Investigation of a Jr(a-) family samples, identification of the mutant and assessment of the differences of Jr antigen density of the Jr(a-) family members, random adult and newborn individuals' RBCs. BACKGROUND: The anti-Jra antibody is generated when a Jr(a-) individual pregnant or transfused with Jr(a+) blood unit, which can lead to mild-to-moderate hemolytic disease of the foetus and newborn (HDFN) or hemolytic transfusion reaction (HTR). Several mutations had been identified. The anti-Jra caused HDFN is not rare in East Asia, but due to the lack of antibody and molecular background, it is likely to lead missed detection. METHODS AND MATERIALS: One G4P1 woman had been detected as IAT positive during prenatal examination. Suspected as anti-Jra after the laboratory serological testing, the maternal sample was further assessed by molecular analysis. The antigen density was detected by flow cytometry after reacting with anti-Jra serum in family members and the normal individuals. RESULTS: One novel frameshift mutation c.717delC and one previously identified mutation c.706C > T in ABCG2 was identified on proband. The infant haemoglobin(Hb) and bilirubin increased significantly after exchange transfusion and the severe HDFN was relieved. Flow cytometry results showed that the Jra antigens on adult RBCs were significantly less than those on the infant. CONCLUSION: The c.717delC mutation can lead to the shortening of protein ABCG2 in the site of p.Leu307Stop, result in the loss of Jra antigen. The difference in antigen density between adult and infant RBCs may be a possible reason that leads to severe HDFN but not transfusion reaction. Breastfeeding may lead to slower recovery from HDFN.

The First Genome-Wide Survey of Shortbelly Eel (Dysomma anguillare Barnard, 1923) to Provide Genomic Characteristics, Microsatellite Markers and Complete Mitogenome Information.

Dysomma anguillare is a demersal eel widespread distributing in tropical waters of the Indo-West Pacific and Atlantic. As an important component of the coastal fishery and marine ecosystem, the lack of genomic information for this species severely restricts the progress of relevant researches. In this study, the abecedarian genome-wide characteristics and phylogenetic relationships analyses were carried out based on next-generation sequencing (NGS) platform. The revised genome size was approximately 1 310 Mb, with the largest scaffold length reaching 23 878 bp through K-mer (K = 17) method. The heterozygosity, repetitive rate and average GC content were about 0.94%, 51.93% and 42.23%, respectively. A total of 1 160 104 microsatellite motifs were identified from the de novo assembled genome of D. anguillare, in which dinucleotide repeats accounted for the largest proportion (592 234, 51.05%), the highest occurrence frequency (14.58%) as well as the largest relative abundance (379.27/Mb). The high-polymorphic and moderate-polymorphic loci composed around 73% of the total single sequence repeats (SSRs), showing a latent capacity for subsequent population genetic structure and genetic diversity appraisal researches. Another byproduct of whole-genome sequencing, the double-stranded and circular mitogenome (16 690 bp) was assembled to investigate the evolutionary relationships of D. anguillare. The phylogenic tree constructed with maximum likelihood (ML) method showed that D. anguillare was closely related to Synaphobranchidae species, and the molecular systematic results further supported classical taxonomy status of D. anguillare.

RHD alleles contributing to serologically weak D phenotypes in China: A single-centre study over 10 years.

BACKGROUND AND OBJECTIVES: In cases of serologically weak D phenotypes, RHD genotyping may identify discrepant serotyping results and protect the patient against allogeneic immunization. This study aimed to conduct a comprehensive analysis of weak D alleles in China. MATERIALS AND METHODS: We collected samples carrying weak D antigen during a 10-year period from 2005 to 2014. The intensity and epitopes of D were analysed serologically. Genomic DNA was extracted and used for RHD sequencing and heterozygote analysis. In particular, an in vitro expression method for functional verification of the rare and novel in-frame deletion mutation was developed and then combined with homologous modelling results for analysis. RESULTS: We studied a total of 283 weak D samples from volunteer blood donors and identified 45 RHD alleles among them, 11 of which were reported for the first time. Ten (3.5%) samples surprisingly carried DEL allelic variants and as many as 40 (14.1%) carried the wild-type RHD genotype. Combination of the results of functional experiments and in silico analysis suggested that the rare in-frame deletion mutation may reduce the expression of D antigen by affecting the RhD protein structure. CONCLUSIONS: This study provides an enhanced overview of the distribution characteristics of RHD alleles in Chinese subjects with serologically weak D. An in vitro method to predict the biological significance of variant RHD alleles was also provided. We found inconsistent genotyping and phenotypic results in some samples, indicating the existence of additional regulatory mechanisms.

Development of novel models for predicting mismatch repair protein deficiency and relevant disease-free survival in colorectal cancer patients.

PURPOSE: DNA mismatch repair (MMR) protein deficiency has attached more attention for its potential to be a biomarker of immunotherapy for colorectal cancer (CRC) patients. However, clinical models involving the expression status of MMR protein are rare. Herein, we sought to develop two clinical models (a diagnostic model for the prediction of MMR status and a prognostic model for the prediction of disease-free survival) for CRC patients. METHODS: A total of 582 CRC patients were finally included. There were 53 patients with deficient expression of MMR protein. The differences between the deficient MMR (dMMR) group and the proficient MMR (pMMR) group were analyzed. RESULTS: Compared to pMMR patients, those with dMMR status were younger and had better pathological features (depth of invasion, lymph node metastasis, distant metastasis, pathological stage, perineuronal invasion, and PLT level) and disease-free survival (DFS). The tumor location of the left colon, adenocarcinoma, and abnormal PLT level were identified as the independent predictors for pMMR. Based on these data, we developed the diagnostic model using Logistic regression analysis. It showed a satisfactory accuracy (AUC = 82.3% in the derivate set; AUC = 73.6% in the validation set). Furthermore, pMMR, poorer differentiation, perineuronal invasion, distant metastasis, lower hemoglobin level, and abnormal CEA level were established as the independent prognostic factors of poorer DFS. Based on them, a prognostic model with valuable performance (1-year AUC = 75.5%/3-year AUC = 76.9% in the derivate set; 1-year AUC = 72.3%/3-year AUC = 73.8% in the validation set) was developed. CONCLUSIONS: Our diagnostic and prognostic models could identify CRC patients at risk for pMMR protein expression and disease recurrence. It may contribute to improving the diagnosis and treatment of CRC patients at an individual level.

Improving pre-harvest sprouting resistance in rice by editing OsABA8ox using CRISPR/Cas9.

KEY MESSAGE: Knock out OsABA8ox helps improve pre-harvest spouting resistance and do not affect rice yield. Pre-harvest sprouting(PHS) is a phenomenon that the seeds of crops germinate preharvest, which reduces the yield and quality of rice. Abscisic acid(ABA) is one of the phytohormones that promotes seed dormancy. ABA8' hydroxylase is the main enzyme that can catabolism ABA in plant. There are three genes that encode ABA8' hydroxylase in rice, named OsABA8ox1, OsABA8ox2 and OsABA8ox3. In this study, we use CRISPR/Cas9 gene editing technology to target these three genes in Ningjing6 and find that the knockout transgenic lines are all significantly strengthen in seed dormancy and have no effect on the yield. By a series of quantitative experiments, we consider that after knock out OsABA8ox, the high endogenous ABA level will influence the ABA signal which suppress the substantial and energy metabolism in the seeds, and finally led to higher dormancy.

Ribonuclease H-like gene SMALL GRAIN2 regulates grain size in rice through brassinosteroid signaling pathway.

Grain size is a key agronomic trait that determines the yield in plants. Regulation of grain size by brassinosteroids (BRs) in rice has been widely reported. However, the relationship between the BR signaling pathway and grain size still requires further study. Here, we isolated a rice mutant, named small grain2 (sg2), which displayed smaller grain and a semi-dwarf phenotype. The decreased grain size was caused by repressed cell expansion in spikelet hulls of the sg2 mutant. Using map-based cloning combined with a MutMap approach, we cloned SG2, which encodes a plant-specific protein with a ribonuclease H-like domain. SG2 is a positive regulator downstream of GLYCOGEN SYNTHASE KINASE2 (GSK2) in response to BR signaling, and its mutation causes insensitivity to exogenous BR treatment. Genetical and biochemical analysis showed that GSK2 interacts with and phosphorylates SG2. We further found that BRs enhance the accumulation of SG2 in the nucleus, and subcellular distribution of SG2 is regulated by GSK2 kinase activity. In addition, Oryza sativa OVATE family protein 19 (OsOFP19), a negative regulator of grain shape, interacts with SG2 and plays an antagonistic role with SG2 in controlling gene expression and grain size. Our results indicated that SG2 is a new component of GSK2-related BR signaling response and regulates grain size by interacting with OsOFP19.

Correlated Insulating States and Transport Signature of Superconductivity in Twisted Trilayer Graphene Superlattices.

Layers of two-dimensional materials stacked with a small twist angle give rise to beating periodic patterns on a scale much larger than the original lattice, referred to as a "moiré superlattice." Here, we demonstrate a higher-order "moiré of moiré" superlattice in twisted trilayer graphene with two consecutive small twist angles. We report correlated insulating states near the half filling of the moiré of moiré superlattice at an extremely low carrier density (∼10^{10} cm^{-2}), near which we also report a zero-resistance transport behavior typically expected in a 2D superconductor. The full-occupancy (ν=-4 and ν=4) states are semimetallic and gapless, distinct from the twisted bilayer systems.

Ultraheavy and Ultrarelativistic Dirac Quasiparticles in Sandwiched Graphenes.

Electrons in quantum materials exhibiting coexistence of dispersionless (flat) bands piercing dispersive (steep) bands give rise to strongly correlated phenomena and are associated with unconventional superconductivity. We show that in twisted sandwiched graphene (TSWG)-a three-layer van der Waals heterostructure with a twisted middle layer-steep Dirac cones can coexist with dramatic band flattening at the same energy scale, if twisted by 1.5°. This phenomenon is not stable in the simplified continuum models. The key result of this Letter is that the flat bands become stable only as a consequence of lattice relaxation processes included in our atomistic calculations. Further on, external fields can change the relative energy offset between the Dirac cone vertex and the flat bands and enhance band hybridization, which could permit controlling correlated phases. Our work establishes twisted sandwiched graphene as a new platform for research into strongly interacting two-dimensional quantum matter.

Twisted Trilayer Graphene: A Precisely Tunable Platform for Correlated Electrons.

We introduce twisted trilayer graphene (tTLG) with two independent twist angles as an ideal system for the precise tuning of the electronic interlayer coupling to maximize the effect of correlated behaviors. As established by experiment and theory in the related twisted bilayer graphene system, van Hove singularities (VHS) in the density of states can be used as a proxy of the tendency for correlated behaviors. To explore the evolution of VHS in the twist-angle phase space of tTLG, we present a general low-energy electronic structure model for any pair of twist angles. We show that the basis of the model has infinite dimensions even at a finite energy cutoff and that no Brillouin zone exists even in the continuum limit. Using this model, we demonstrate that the tTLG system exhibits a wide range of magic angles at which VHS merge and that the density of states has a sharp peak at the charge-neutrality point through two distinct mechanisms: the incommensurate perturbation of twisted bilayer graphene's flatbands or the equal hybridization between two bilayer moiré superlattices.