Digital microfluidics-based digital counting of single-cell copy number variation (dd-scCNV Seq).

Single-cell copy number variations (CNVs), major dynamic changes in humans, result in differential levels of gene expression and account for adaptive traits or underlying disease. Single-cell sequencing is needed to reveal these CNVs but has been hindered by single-cell whole-genome amplification (scWGA) bias, leading to inaccurate gene copy number counting. In addition, most of the current scWGA methods are labor intensive, time-consuming, and expensive with limited wide application. Here, we report a unique single-cell whole-genome library preparation approach based on digital microfluidics for digital counting of single-cell Copy Number Variation (dd-scCNV Seq). dd-scCNV Seq directly fragments the original single-cell DNA and uses these fragments as templates for amplification. These reduplicative fragments can be filtered computationally to generate the original partitioned unique identified fragments, thereby enabling digital counting of copy number variation. dd-scCNV Seq showed an increase in uniformity in the single-molecule data, leading to more accurate CNV patterns compared to other methods with low-depth sequencing. Benefiting from digital microfluidics, dd-scCNV Seq allows automated liquid handling, precise single-cell isolation, and high-efficiency and low-cost genome library preparation. dd-scCNV Seq will accelerate biological discovery by enabling accurate profiling of copy number variations at single-cell resolution.

A calmodulin-gated calcium channel links pathogen patterns to plant immunity.

Pathogen-associated molecular patterns (PAMPs) activate innate immunity in both animals and plants. Although calcium has long been recognized as an essential signal for PAMP-triggered immunity in plants, the mechanism of PAMP-induced calcium signalling remains unknown1,2. Here we report that calcium nutrient status is critical for calcium-dependent PAMP-triggered immunity in plants. When calcium supply is sufficient, two genes that encode cyclic nucleotide-gated channel (CNGC) proteins, CNGC2 and CNGC4, are essential for PAMP-induced calcium signalling in Arabidopsis3-7. In a reconstitution system, we find that the CNGC2 and CNGC4 proteins together-but neither alone-assemble into a functional calcium channel that is blocked by calmodulin in the resting state. Upon pathogen attack, the channel is phosphorylated and activated by the effector kinase BOTRYTIS-INDUCED KINASE1 (BIK1) of the pattern-recognition receptor complex, and this triggers an increase in the concentration of cytosolic calcium8-10. The CNGC-mediated calcium entry thus provides a critical link between the pattern-recognition receptor complex and calcium-dependent immunity programs in the PAMP-triggered immunity signalling pathway in plants.

Fluid nanoporous microinterface enables multiscale-enhanced affinity interaction for tumor-derived extracellular vesicle detection.

Tumor-derived extracellular vesicles (T-EVs) represent valuable markers for tumor diagnosis and treatment guidance. However, nanoscale sizes and the low abundance of marker proteins of T-EVs restrict interfacial affinity reaction, leading to low isolation efficiency and detection sensitivity. Here, we engineer a fluid nanoporous microinterface (FluidporeFace) in a microfluidic chip by decorating supported lipid bilayers (SLBs) on nanoporous herringbone microstructures with a multiscale-enhanced affinity reaction for efficient isolation of T-EVs. At the microscale level, the herringbone micropattern promotes the mass transfer of T-EVs to the surface. At the nanoscale level, nanoporousity can overcome boundary effects for close contact between T-EVs and the interface. At the molecular level, fluid SLBs afford clustering of recognition molecules at the binding site, enabling multivalent binding with an ∼83-fold increase of affinity compared with the nonfluid interface. With the synergetic enhanced mass transfer, interface contact, and binding affinity, FluidporeFace affords ultrasensitive detection of T-EVs with a limit of detection of 10 T-EVs µL-1, whose PD-L1 expression levels successfully distinguish cancer patients from healthy donors. We expect this multiscale enhanced interfacial reaction strategy will inspire the biosensor design and expand liquid biopsy applications, especially for low-abundant targets in clinical samples.

Encoded Fusion-Mediated MicroRNA Signature Profiling of Tumor-Derived Extracellular Vesicles for Pancreatic Cancer Diagnosis.

MicroRNAs (miRNAs) in tumor-derived extracellular vesicles (tEVs) are important cancer biomarkers for cancer screening and early diagnosis. Multiplex detection of miRNAs in tEVs facilitates accurate diagnosis but remains a challenge. Herein, we propose an encoded fusion strategy to profile the miRNA signature in tEVs for pancreatic cancer diagnosis. A panel of encoded-targeted-fusion beads was fabricated for the selective recognition and fusion of tEVs, with the turn-on fluorescence signals of molecule beacons for miRNA quantification and barcode signals for miRNA identification using readily accessible flow cytometers. Using this strategy, six types of pancreatic-cancer-associated miRNAs can be profiled in tEVs from 2 µL plasma samples (n = 36) in an isolation-free and lysis-free manner with only 2 h of processing, offering a high accuracy (98%) to discriminate pancreatic cancer, pancreatitis, and healthy donors. This encoded fusion strategy exhibits great potential for multiplex profiling of miRNA in tEVs, offering new avenues for cancer diagnosis and screening.

In Vitro Hypoglycemic Diterpenoids from the Roots of Croton yunnanensis.

Fifteen compounds including nine new diterpenes were isolated from the roots of Croton yunnanensis. By HRESIMS, NMR, ECD data, and X-ray diffraction analysis, the new compounds were characterized as eight neo-clerodane diterpenes (compounds 1-8) and one 15,16-dinor-ent-pimarane diterpene (9). All diterpenes were assayed for their hypoglycemic activities. Compounds 1-4, 6, 7, and 10 promoted glucose uptake activity in insulin-resistant 3T3-L1 adipocytes. Compounds 1 and 6 showed insulin sensitizing activity, potentiating conspicuously their glucose uptake activity at a concentration of 20 µM when treated synergistically with low-concentration insulin at 1 nM.

[Analysis of TUBB4A gene variant in a patient with adolescent-onset hypomyelinating leukodystrophy with atrophy of basal ganglia and cerebellum].

OBJECTIVE: To explore the clinical characteristics and genetic etiology of a patient with adolescent-onset hypomyelinated leukodystrophy with atrophy of basal ganglia and cerebellum (H-ABC). METHODS: A patient who was diagnosed with H-ABC in March 2018 at the First Affiliated Hospital of Nanjing Medical University was selected as the study subject. Clinical data was collected. Peripheral venous blood samples of the patient and his parents were collected. The patient was subjected to whole exome sequencing (WES). Candidate variant was verified by Sanger sequencing. RESULTS: The patient, a 31-year-old male, had manifested with developmental retardation, cognitive decline and abnormal gait. WES revealed that he has harbored a heterozygous c.286G>A variant of the TUBB4A gene. Sanger sequencing confirmed that neither of his parents has carried the same variant. Analysis with SIFT online software indicated the amino acid encoded by this variant is highly conserved among various species. This variant has been recorded by the Human Gene Mutation Database (HGMD) with a low population frequency. The 3D structure constructed by PyMOL software showed that the variant has a harmful effect on the structure and function of the protein. According to the guidelines formulated by the American College of Medical Genetics and Genomics (ACMG), the variant was rated as likely pathogenic. CONCLUSION: The c.286G>A (p.Gly96Arg) variant of the TUBB4A gene probably underlay the hypomyelinating leukodystrophy with atrophy of basal ganglia and cerebellum in this patient. Above finding has enriched the spectrum of TUBB4A gene variants and enabled early definitive diagnosis of this disorder.

[Chemical constituents from Salacia polysperma].

Nine compounds were isolated from the 90% ethanol extract of Salacia polysperma by silica gel, Sephadex LH-20 column chromatography, together with preparative HPLC methods. Based on HR-ESI-MS, MS, 1D and 2D NMR spectral analyses, the structures of the nine compounds were identified as 28-hydroxy wilforlide B(1), wilforlide A(2), 1ß,3ß-dihydroxyurs-9(11),12-diene(3),(-)-epicatechin(4),(+)-catechin(5),(-)-4'-O-methyl-ent-galloepicatechin(6), 3-hydroxy-1-(4-hydroxy-3-methoxy-phenyl)propan-1-one(7),(-)-(7S,8R)-4-hydroxy-3,3',5'-trimethoxy-8',9'-dinor-8,4'-oxyneoligna-7,9-diol-7'-aldehyde(8), and vanillic acid(9). Compound 1 is a new oleanane-type triterpene lactone. Compounds 1, 3, 4, 7-9 were isolated from the Salacia genus for the first time. All compounds were assayed for their α-glucosidase inhibitory activity. The results suggested that compound 8 exhibited moderate α-glucosidase inhibitory activity, with an IC_(50) value of 37.2 µmol·L~(-1), and the other compounds showed no α-glucosidase inhibitory activity.

[A new xanthone from hulls of Garcinia mangostana and its cytotoxic activity].

Eight compounds were isolated from ethyl acetate fraction of 80% ethanol extract of the hulls of Garcinia mangostana by silica gel, Sephadex LH-20 column chromatography, as well as prep-HPLC methods. By HR-ESI-MS, MS, 1D and 2D NMR spectral analyses, the structures of the eight compounds were identified as 16-en mangostenone E(1), α-mangostin(2), 1,7-dihydroxy-2-(3-methy-lbut-2-enyl)-3-methoxyxanthone(3), cratoxyxanthone(4), 2,6-dimethoxy-para-benzoquinone(5), methyl orselinate(6), ficusol(7), and 4-(4-carboxy-2-methoxyphenoxy)-3,5-dimethoxybenzoic acid(8). Compound 1 was a new xanthone, and compound 4 was a xanthone dimer, compound 5 was a naphthoquinone. All compounds were isolated from this plant for the first time except compounds 2 and 3. Cytotoxic bioassay suggested that compounds 1, 2 and 4 possessed moderate cytotoxicity, suppressing HeLa cell line with IC_(50) va-lues of 24.3, 35.5 and 17.1 µmol·L~(-1), respectively. Compound 4 also could suppress K562 cells with an IC_(50) value of 39.8 µmol·L~(-1).

ARMOR: Auto-Assembled Resilient Biomimetic Calcified Ornaments for Selective Cell Protection by Dual-Aptamer-Driven Hybridization Chain Reaction.

Unlike plant and microbial cells having cell walls, the outermost layer of mammalian cell is a delicate, two-layered structure of phospholipids with proteins embedded, which is susceptible to environmental changes. It is necessary to create an "armor" on cell surface to protect cell integrity. Here, we propose an Auto-assembled Resilient bioMimetic calcified ORnaments (ARMOR) strategy driven by dual-aptamer-based hybridization chain reaction (HCR) and Ca2+ assisted calcification for selective cell protection. This co-recognition design enhances the selectivity and leverages robust in situ signal amplification by HCR to improve the sensitivity. The calcified shell is cogenerated by crosslinking the alginate-HCR product with Ca2+ ion. ARMOR has high efficiency for shielding cells from environmental assaults, which can be applied to circulating tumor cell (CTC) protection, isolation, and identification, maintaining the native state and intact genetic information for downstream analysis.

Hierarchical Fluid Interface Enables Spatiotemporal Regulation of Ligand Distribution to Increase Kinetics and Thermodynamics of Interfacial Binding Reaction.

In nature, regulation of the spatiotemporal distribution of interfacial receptors and ligands leads to optimum binding kinetics and thermodynamics of receptor-ligand binding reactions within interfaces. Inspired by this, we report a hierarchical fluid interface (HieFluidFace) to regulate the spatiotemporal distribution of interfacial ligands to increase the rate and thermodynamic favorability of interfacial binding reactions. Each aptamer-functionalized gold nanoparticle, termed spherical aptamer (SAPT), is anchored on a supported lipid bilayer without fluidity, like an "island", and is surrounded by many fluorescent aptamers (FAPTs) with free fluidity, like "rafts". Such ligand "island-rafts" model provides a large reactive cross-section for rapid binding to cellular receptors. The synergistic multivalency of SAPTs and FAPTs improves interfacial affinity for tight capture. Moreover, FAPTs accumulate at binding sites to bind to cellular receptors with clustered fluorescence to "lighten" cells for direct identification. Thus, HieFluidFace in a microfluidic chip achieves high-performance capture and identification of circulating tumor cells from clinical samples, providing a new paradigm to optimize the kinetics and thermodynamics of interfacial binding reactions.

A Fluid Multivalent Magnetic Interface for High-Performance Isolation and Proteomic Profiling of Tumor-Derived Extracellular Vesicles.

Isolation and analysis of tumor-derived extracellular vesicles (T-EVs) are important for clinical cancer management. Here, we develop a fluid multivalent magnetic interface (FluidmagFace) in a microfluidic chip for high-performance isolation, release, and protein profiling of T-EVs. The FluidmagFace increases affinity by 105-fold with fluidity-enhanced multivalent binding to improve isolation efficiency by 13.9 % compared with a non-fluid interface. Its anti-adsorption property and microfluidic hydrodynamic shear minimize contamination, increasing detection sensitivity by two orders of magnitude. Moreover, its reversibility and expandability allow high-throughput recovery of T-EVs for mass spectrometric protein analysis. With the chip, T-EVs were detected in all tested cancer samples with identification of differentially expressed proteins compared with healthy controls. The FluidmagFace opens a new avenue to isolation and release of targets for cancer diagnosis and biomarker discovery.

[Advance in research on pathogenetic genes for amyotrophic lateral sclerosis].

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease which is associated with genetic and environmental factors, though the pathogenesis is still unclear and there is also a lack of effective treatment. With the rapid advance of genetic testing techniques, over 30 genes have been associated with the disease. Some ALS patients harboring genetic variants may present unique clinical characteristics and particular mode of inheritance, but the correlation between genotype and phenotype is still not very clear. Studies have shown that research on the pathogenic genes of ALS is important for the diagnosis and selection of potential drug targets. Here the pathogenic genes of ALS, in particular the newly discovered genes, and their underlying mechanisms are reviewed. The necessity of genetic testing for ALS patients is also stressed.

A chloride efflux transporter, BIG RICE GRAIN 1, is involved in mediating grain size and salt tolerance in rice.

Grain size is determined by the size and number of cells in the grain. The regulation of grain size is crucial for improving crop yield; however, the genes and molecular mechanisms that control grain size remain elusive. Here, we report that a member of the detoxification efflux carrier /Multidrug and Toxic Compound Extrusion (DTX/MATE) family transporters, BIG RICE GRAIN 1 (BIRG1), negatively influences grain size in rice (Oryza sativa L.). BIRG1 is highly expressed in reproductive organs and roots. In birg1 grain, the outer parenchyma layer cells of spikelet hulls are larger than in wild-type (WT) grains, but the cell number is unaltered. When expressed in Xenopus laevis oocytes, BIRG1 exhibits chloride efflux activity. Consistent with this role of BIRG1, the birg1 mutant shows reduced tolerance to salt stress at a toxic chloride level. Moreover, grains from birg1 plants contain a higher level of chloride than those of WT plants when grown under normal paddy field conditions, and the roots of birg1 accumulate more chloride than those of WT under saline conditions. Collectively, the data suggest that BIRG1 in rice functions as a chloride efflux transporter that is involved in mediating grain size and salt tolerance by controlling chloride homeostasis.

Dehydration stress extends mRNA 3' untranslated regions with noncoding RNA functions in Arabidopsis.

The 3' untranslated regions (3' UTRs) of mRNAs play important roles in the regulation of mRNA localization, translation, and stability. Alternative cleavage and polyadenylation (APA) generates mRNAs with different 3' UTRs, but the involvement of this process in stress response has not yet been clarified. Here, we report that a subset of stress-related genes exhibits 3' UTR extensions of their mRNAs during dehydration stress. These extended 3' UTRs have characteristics of long noncoding RNAs and likely do not interact with miRNAs. Functional studies using T-DNA insertion mutants reveal that they can act as antisense transcripts to repress expression levels of sense genes from the opposite strand or can activate the transcription or lead to read-through transcription of their downstream genes. Further analysis suggests that transcripts with 3' UTR extensions have weaker poly(A) signals than those without 3' UTR extensions. Finally, we show that their biogenesis is partially dependent on a trans-acting factor FPA. Taken together, we report that dehydration stress could induce transcript 3' UTR extensions and elucidate a novel function for these stress-induced 3' UTR extensions as long noncoding RNAs in the regulation of their neighboring genes.

Modification of phytosterol composition influences cotton fiber cell elongation and secondary cell wall deposition.

BACKGROUND: Cotton fiber is a single cell that arises from the epidermis of ovule. It is not only a main economic product of cotton, but an ideal material for studying on the growth and development of plant cell. Our previous study indicated that phytosterol content and the ratio of campesterol to sitosterol fluctuated regularly in cotton fiber development. However, what effects of modified phytosterol content and composition on the growth and development of cotton fiber cell is unknown. In this study, we overexpressed the GhSMT2-1, a cotton homologue of sterol C-24 methyltransferase 2 gene in transgenic upland cotton plants to modify phytosterol content and composition in fiber cells and investigated the changes on fiber elongation and secondary cell wall deposition. RESULTS: GhSMT2-1 overexpression led to changes of phytosterol content and the ratio of campesterol to sitosterol in fiber cell. At the rapid elongation stage of fiber cell, total phytosterol and sitosterol contents were increased while campesterol content was decreased in transgenic fibers when compared to control fibers. Accordingly, the ratio of campesterol to sitosterol declined strikingly. Simultaneously, the transgenic fibers were shorter and thicker than control fibers. Exogenous application of sitosterol or campesterol separately inhibited control fiber cell elongation in cotton ovule culture system in vitro. In addition, campesterol treatment partially rescued transgenic fiber elongation. CONCLUSION: These results elucidated that modification of phytosterol content and composition influenced fiber cell elongation and secondary cell wall formation. High sitosterol or low ratio of campesterol to sitosterol suppresses fiber elongation and/or promote secondary cell wall deposition. The roles of sitosterol and campesterol were discussed in fiber cell development. There might be a specific ratio of campesterol to sitosterol in different developmental stage of cotton fibers, in which GhSMT2-1 play an important role. Our study, at a certain degree, provides novel insights into the regulatory mechanisms of fiber cell development.

A novel WARS mutation (p.Asp314Gly) identified in a Chinese distal hereditary motor neuropathy family.

Distal hereditary motor neuropathy (dHMN) is a clinically and genetically heterogeneous group of inherited neuropathies characterized by distal limb muscle wasting and weakness with no or minimal sensory abnormalities. To investigate the clinical and genetic features of dHMN caused by WARS mutations in mainland China, we performed Sanger sequencing of the coding and untranslated region (UTR) regions of WARS in 160 unresolved dHMN and Charcot-Marie-Tooth (CMT) index patients. We detected a novel heterozygous variant c.941A>G (p.Asp314Gly) of WARS in an index patient from an autosomal dominant dHMN family including five affected members over three generations. The variant completely co-segregates with the dHMN phenotype in the family, and it was classified as likely pathogenic according to the American College of Medical Genetics and Genomics standards and guidelines. The clinical features included juvenile to adult onset (15-23 years), distal wasting and weakness, minimal sensory disturbance and length-dependent motor axonal degeneration with CMT examination score ranging from 6 to 10. Our report further confirms the role of WARS in dHMN and indicates that the variant c.941A>G (p.Asp314Gly) of WARS is related to a mild to moderate affected and later onset phenotype of dHMN.

Microbial Targeted Degradation Pretreatment: A Novel Approach to Preparation of Activated Carbon with Specific Hierarchical Porous Structures, High Surface Areas, and Satisfactory Toluene Adsorption Performance.

Hierarchical porous carbon shows great potential for volatile organic compounds (VOCs) removal due to its high surface area and abundant porous framework. However, current fabrication protocols are complex and cause secondary pollution, limiting their application. Here, as a novel strategy, microbial lignocellulose decomposition as a pretreatment was introduced to fabricate hierarchical porous carbon (M-AC) from crude biomass substrate. The M-AC samples had high specific surface areas (maximum: 2290 m2·g-1) and surfaces characterized by needle-like protrusions with a high degree of disorder attributed to hierarchical porous structures. Dynamic toluene adsorption indicated that the carbon materials with microbial pretreatment had much better adsorption performances (maximum: 446 mg/g) than activated carbon without pretreatment. The M-AC material pretreated with a cellulose-degrading microbe showed the best adsorption capacity due to well-developed micropores, whereas the M-AC material pretreated with a lignin-degrading microbe showed excellent transport diffusion due to well-developed mesopores. Therefore, this simple and effective approach using microbial decomposition pretreatment is promising for the development of hierarchical porous carbons with adjustable pore structures and high specific surface areas to remove target VOCs in practical applications.

A noncoding RNA transcribed from the AGAMOUS (AG) second intron binds to CURLY LEAF and represses AG expression in leaves.

Dispersed H3K27 trimethylation (H3K27me3) of the AGAMOUS (AG) genomic locus is mediated by CURLY LEAF (CLF), a component of the Polycomb Repressive Complex (PRC) 2. Previous reports have shown that the AG second intron, which confers AG tissue-specific expression, harbors sequences targeted by several positive and negative regulators. Using RACE reverse transcription polymerase chain reaction, we found that the AG intron 2 encodes several noncoding RNAs. RNAi experiment showed that incRNA4 is needed for CLF repressive activity. AG-incRNA4RNAi lines showed increased leaf AG mRNA levels associated with a decrease of H3K27me3 levels; these plants displayed AG overexpression phenotypes. Genetic and biochemical analyses demonstrated that the AG-incRNA4 can associate with CLF to repress AG expression in leaf tissues through H3K27me3-mediated repression and to autoregulate its own expression level. The mechanism of AG-incRNA4-mediated repression may be relevant to investigations on tissue-specific expression of Arabidopsis MADS-box genes.

Regulated intramembrane proteolysis of the AXL receptor kinase generates an intracellular domain that localizes in the nucleus of cancer cells.

Deregulation of the TAM (TYRO3, AXL, and MERTK) family of receptor tyrosine kinases (RTKs) has recently been demonstrated to predominately promote survival and chemoresistance of cancer cells. Intramembrane proteolysis mediated by presenilin/γ-secretase is known to regulate the homeostasis of some RTKs. In the present study, we demonstrate that AXL, but not TYRO3 or MERTK, is efficiently and sequentially cleaved by α- and γ-secretases in various types of cancer cell lines. Proteolytic processing of AXL redirected signaling toward a secretase-mediated pathway, away from the classic, well-known, ligand-dependent canonical RTK signaling pathway. The AXL intracellular domain cleavage product, but not full-length AXL, was further shown to translocate into the nucleus via a nuclear localization sequence that harbored a basic HRRKK motif. Of interest, we found that the γ-secretase-uncleavable AXL mutant caused an elevated chemoresistance in non-small-cell lung cancer cells. Altogether, our findings suggest that AXL can undergo sequential processing mediated by various proteases kept in a homeostatic balance. This newly discovered post-translational processing of AXL may provide an explanation for the diverse functions of AXL, especially in the context of drug resistance in cancer cells.-Lu, Y., Wan, J., Yang, Z., Lei, X., Niu, Q., Jiang, L., Passtoors, W. M., Zang, A., Fraering, P. C., Wu, F. Regulated intramembrane proteolysis of the AXL receptor kinase generates an intracellular domain that localizes in the nucleus of cancer cells.

Facile On-Site Aqueous Pollutant Monitoring Using a Flexible, Ultralight, and Robust Surface-Enhanced Raman Spectroscopy Substrate: Interface Self-Assembly of Au@Ag Nanocubes on a Polyvinyl Chloride Template.

Aquatic ecosystems and human health have been seriously threatened by illegal discharge of wastewater, while simple and effective monitoring methods are still sparse. Here, we propose a facile method for on-site pollutant monitoring by surface-enhanced Raman spectroscopy with a novel substrate. This substrate is fabricated by interface self-assembly of Au@Ag nanocubes (NCs) on a simultaneously formed polyvinyl chloride (PVC) template, followed by coating with a thin Au film. The Au@Ag@Au-NCs/PVC film is flexible, ultralight, and robust and could float on the surface of water and firmly contact with water even under harsh environmental conditions. Moreover, the Au@Ag@Au-NCs/PVC film is translucent, allowing penetration of laser beams and enhancement of Raman signals. When thiram was used as a model contaminant in aqueous solution, a good linear relationship ( R2 = 0.972) was obtained over the range of 0.1-50 ppb with a detection limit of 0.1 ppb. Raman signals of thiram can be instantly and consecutively detected with the enhancement of the film in the simulated experiments, suggesting its possible use in the long run. Furthermore, the film can be easily regenerated by NaBH4 solution washing, which could reduce the operating cost. In summary, the Au@Ag@Au-NCs/PVC film has great potential in on-site pollutant monitoring in aqueous environments with a portable Raman spectrometer.