Progress in the development of modulators targeting Frizzleds.

The Frizzleds (FZDs) receptors on the cell surface belong to the class F of G protein-coupled receptors (GPCRs) which are the major receptors of WNT protein that mediates the classical WNT signaling pathway and other non-classical pathways. Besides, the FZDs also play a core role in tissue regeneration and tumor occurrence. With the structure and mechanism of FZDs activation becoming clearer, a series of FZDs modulators (inhibitors and agonists) have been developed, with the hope of bringing benefits to the treatment of cancer and degenerative diseases. Most of the FZDs inhibitors (small molecules, antibodies or designed protein inhibitors) block WNT signaling through binding to the cysteine-rich domain (CRD) of FZDs. Several small molecules impede FZDs activation by targeting to the third intracellular domain or the transmembrane domain of FZDs. However, three small molecules (FZM1.8, SAG1.3 and purmorphamine) activate the FZDs through direct interaction with the transmembrane domain. Another type of FZDs agonists are bivalent or tetravalent antibodies which activate the WNT signaling via inducing FZD-LRP5/6 heterodimerization. In this article, we reviewed the FZDs modulators reported in recent years, summarized the critical molecules' discovery processes and the elucidated relevant structural and pharmacological mechanisms. We believe the summaried molecular mechanisms of the relevant modulators could provide important guidance and reference for the future development of FZD modulators.

Novel truncating variants in CTNNB1 cause familial exudative vitreoretinopathy.

BACKGROUND: Familial exudative vitreoretinopathy (FEVR) is an inheritable blinding disorder with clinical and genetic heterogeneity. Heterozygous variants in the CTNNB1 gene have been reported to cause FEVR. However, the pathogenic basis of CTNNB1-associated FEVR has not been fully explored. METHODS: Whole-exome sequencing was performed on the genomic DNA of probands. Dual-luciferase reporter assay, western blotting and co-immunoprecipitation were used to characterise the impacts of variants. Quantitative real-time PCR, EdU (5-ethynyl-2'-deoxyuridine) incorporation assay and immunocytochemistry were performed on the primary human retinal microvascular endothelial cells (HRECs) to investigate the effect of CTNNB1 depletion on the downstream genes involved in Norrin/ß-catenin signalling, cell proliferation and junctional integrity, respectively. Transendothelial electrical resistance assay was applied to measure endothelial permeability. Heterozygous endothelial-specific Ctnnb1-knockout mouse mice were generated to verify FEVR-like phenotypes in the retina. RESULTS: We identified two novel heterozygous variants (p.Leu103Ter and p.Val199LeufsTer11) and one previously reported heterozygous variant (p.His369ThrfsTer2) in the CTNNB1 gene. These variants caused truncation and degradation of ß-catenin that reduced Norrin/ß-catenin signalling activity. Additionally, knockdown (KD) of CTNNB1 in HRECs led to diminished mRNA levels of Norrin/ß-catenin targeted genes, reduced cell proliferation and compromised junctional integrity. The Cre-mediated heterozygous deletion of Ctnnb1 in mouse endothelial cells (ECs) resulted in FEVR-like phenotypes. Moreover, LiCl treatment partially rescued the defects in CTNNB1-KD HRECs and EC-specific Ctnnb1 heterozygous knockout mice. CONCLUSION: Our findings reinforced the current pathogenesis of Norrin/ß-catenin for FEVR and expanded the causative variant spectrum of CTNNB1 for the prenatal diagnosis and genetic counselling of FEVR.

A comprehensive functional analysis on the pathogenesis of novel TSPAN12 and NDP variants in familial exudative vitreoretinopathy.

Familial exudative vitreoretinopathy (FEVR) is an inherited blinding disorder; however, the known FEVR-associated variants account for approximately only 50% cases. Currently, the pathogenesis of most reported variants is not well studied, we aim to identify novel variants from FEVR-associated genes and perform a comprehensive functional analysis to uncover the pathogenesis of variants that cause FEVR. Using targeted gene panel and Sanger sequencing, we identified six novel and three known variants in TSPAN12 and NDP. These variants were demonstrated to cause significant inhibition of Norrin/ß-catenin pathway by dual-luciferase reporter assay and western blot analysis. Structural analysis and co-immunoprecipitation revealed compromised interactions between missense variants and binding partners in the Norrin/ß-catenin pathway. Immunofluorescence and subcellular protein extraction were performed to reveal the abnormal subcellular trafficking. Additionally, over-expression of TSPAN12 successfully enhanced the Norrin/ß-catenin signaling activity by strengthening the binding affinity of mutant Norrin with FZD4 or LRP5. Together, these observations expanded the spectrum of FEVR-associated variants for the genetic counseling and prenatal diagnosis of FEVR, as well providing a potential therapeutic strategy for the treatment of FEVR.

NONSTOP GLUMES1 Encodes a C2H2 Zinc Finger Protein That Regulates Spikelet Development in Rice.

The spikelet is an inflorescence structure unique to grasses. The molecular mechanisms underlying spikelet development and evolution are unclear. In this study, we characterized three allelic recessive mutants in rice (Oryza sativa): nonstop glumes 1-1 (nsg1-1), nsg1-2, and nsg1-3 In these mutants, organs such as the rudimentary glume, sterile lemma, palea, lodicule, and filament were elongated and/or widened, or transformed into lemma- and/or marginal region of the palea-like organs. NSG1 encoded a member of the C2H2 zinc finger protein family and was expressed mainly in the organ primordia of the spikelet. In the nsg1-1 mutant spikelet, LHS1 DL, and MFO1 were ectopically expressed in two or more organs, including the rudimentary glume, sterile lemma, palea, lodicule, and stamen, whereas G1 was downregulated in the rudimentary glume and sterile lemma. Furthermore, the NSG1 protein was able to bind to regulatory regions of LHS1 and then recruit the corepressor TOPLESS-RELATED PROTEIN to repress expression by downregulating histone acetylation levels of the chromatin. The results suggest that NSG1 plays a pivotal role in maintaining organ identities in the spikelet by repressing the expression of LHS1, DL, and MFO1.

Dysregulated m6A modification promotes lipogenesis and development of non-alcoholic fatty liver disease and hepatocellular carcinoma.

Type 2 diabetes mellitus (DM2) is associated closely with non-alcoholic fatty liver disease (NAFLD) by affecting lipid metabolism, which may lead to non-alcoholic steatohepatitis (NASH), fibrosis, and hepatocellular carcinoma (HCC). N6-methyladenosine (m6A) RNA methylation is an important epigenetic regulation for gene expression and is related to HCC development. We developed a new NAFLD model oriented from DM2 mouse, which spontaneously progressed to histological features of NASH, fibrosis, and HCC with high incidence. By RNA sequencing, protein expression and methylated RNA immunoprecipitation (MeRIP)-qPCR analysis, we found that enhanced expression of ACLY and SCD1 in this NAFLD model and human HCC samples was due to excessive m6A modification, but not elevation of mature SREBP1. Moreover, targeting METTL3/14 in vitro increases protein level of ACLY and SCD1 as well as triglyceride and cholesterol production and accumulation of lipid droplets. m6A sequencing analysis revealed that overexpressed METTL14 binds to mRNA of ACLY and SCD1 and alters their expression pattern. Our findings demonstrate a new NAFLD mouse model that provides a study platform for DM2-related NAFLD and reveals a unique epitranscriptional regulating mechanism for lipid metabolism via m6A-modified protein expression of ACLY and SCD1.

Transcriptome and metabolome analyses reveal the interweaving of immune response and metabolic regulation in pelvic organ prolapse.

INTRODUCTION AND HYPOTHESIS: The pathogenesis of pelvic organ prolapse (POP) remains unknown. Herein, we aim to reveal the molecular profile of POP by transcriptomic and metabolomic analysis. METHODS: We selected 12 samples of uterosacral ligaments (USLs) from 6 POP patients and 6 controls for transcriptomic and metabolomic analyses. Differentially expressed genes (DEGs) were identified using the R package edgeR. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using clusterProfiler, and a protein-protein interaction (PPI) network was constructed using STRING and visualized in Cytoscape. Metabolomic profiling was performed by a liquid chromatography-tandem mass spectrometry system. RESULTS: Transcriptomic analysis identified 487 DEGs between the POP and control groups. Functional enrichment analysis revealed that they were mostly related to immune response terms, including "adaptive immune response," "T cell differentiation," and "T cell activation." In addition, PTPRC, LCK, CD247, IL2RB, CD2, CXR5, JUN, CD3E, IL2RG, and PRF1 were the 10 nodes with the highest node degrees in the PPI network. Metabolomic profiling revealed 290 differentially expressed metabolites, which significantly enriched in "glycerophospholipid metabolism," "nicotinate and nicotinamide metabolism," "glycine, serine, and threonine metabolism," "arginine and proline metabolism," "pyrimidine metabolism," and "purine metabolism." Finally, integrated analysis revealed that the DEGs involved in these significantly enriched metabolic pathways included NT5C1A, GMPR, SDS, ALAS2, CARNS1, PYCR1, P4HA3, PGS1, and NMRK2. CONCLUSIONS: Our findings demonstrate that the immune response and metabolic regulatory pathways are intertwined in POP and might provide new therapeutic targets.

Dysfunction of VIPR2 leads to myopia in humans and mice.

BACKGROUND: Myopia is the leading cause of refractive errors. As its pathogenesis is poorly understood, we determined if the retinal VIP-VIPR2 signalling pathway axis has a role in controlling signalling output that affects myopia development in mice. METHODS: Association analysis meta-study, single-cell transcriptome, bulk RNA sequencing, pharmacological manipulation and VIPR2 gene knockout studies were used to clarify if changes in the VIP-VIPR2 signalling pathway affect refractive development in mice. RESULTS: The SNP rs6979985 of the VIPR2 gene was associated with high myopia in a Chinese Han cohort (randomceffect model: p=0.013). After either 1 or 2 days' form deprivation (FD) retinal VIP mRNA expression was downregulated. Retinal single-cell transcriptome sequencing showed that VIPR2 was expressed mainly by bipolar cells. Furthermore, the cAMP signalling pathway axis was inhibited in some VIPR2+ clusters after 2 days of FD. The selective VIPR2 antagonist PG99-465 induced relative myopia, whereas the selective VIPR2 agonist Ro25-1553 inhibited this response. In Vipr2 knockout (Vipr2-KO) mice, refraction was significantly shifted towards myopia (p<0.05). The amplitudes of the bipolar cell derived b-waves in 7-week-old Vipr2-KO mice were significantly larger than those in their WT littermates (p<0.05). CONCLUSIONS: Loss of VIPR2 function likely compromises bipolar cell function based on presumed changes in signal transduction due to altered signature electrical wave activity output in these mice. As these effects correspond with increases in form deprivation myopia (FDM), the VIP-VIPR2 signalling pathway axis is a viable novel target to control the development of this condition.

Detection of Missing Bolts for Engineering Structures in Natural Environment Using Machine Vision and Deep Learning.

The development of an accurate and efficient method for detecting missing bolts in engineering structures is crucial. To this end, a missing bolt detection method that leveraged machine vision and deep learning was developed. First, a comprehensive dataset of bolt images captured under natural conditions was constructed, which improved the generality and recognition accuracy of the trained bolt target detection model. Second, three deep learning network models, namely, YOLOv4, YOLOv5s, and YOLOXs, were compared, and YOLOv5s was selected as the bolt target detection model. With YOLOv5s as the target recognition model, the bolt head and bolt nut had average precisions of 0.93 and 0.903, respectively. Third, a missing bolt detection method based on perspective transformation and IoU was presented and validated under laboratory conditions. Finally, the proposed method was applied to an actual footbridge structure to test its feasibility and effectiveness in real engineering scenarios. The experimental results showed that the proposed method could accurately identify bolt targets with a confidence level of over 80% and detect missing bolts under different image distances, perspective angles, light intensities, and image resolutions. Moreover, the experimental results on a footbridge demonstrated that the proposed method could reliably detect the missing bolt even at a shooting distance of 1 m. The proposed method provided a low-cost, efficient, and automated technical solution for the safety management of bolted connection components in engineering structures.

A Unique G-Quadruplex Aptamer: A Novel Approach for Cancer Cell Recognition, Cell Membrane Visualization, and RSV Infection Detection.

Surface staining has emerged as a rapid technique for applying external stains to trace cellular identities in diverse populations. In this study, we developed a distinctive aptamer with selective binding to cell surface nucleolin (NCL), bypassing cytoplasmic internalization. Conjugation of the aptamer with a FAM group facilitated NCL visualization on live cell surfaces with laser confocal microscopy. To validate the aptamer-NCL interaction, we employed various methods, including the surface plasmon resonance, IHC-based flow cytometry, and electrophoretic mobility shift assay. The G-quadruplex formations created by aptamers were confirmed with a nuclear magnetic resonance and an electrophoretic mobility shift assay utilizing BG4, a G-quadruplex-specific antibody. Furthermore, the aptamer exhibited discriminatory potential in distinguishing between cancerous and normal cells using flow cytometry. Notably, it functioned as a dynamic probe, allowing real-time monitoring of heightened NCL expression triggered by a respiratory syncytial virus (RSV) on normal cell surfaces. This effect was subsequently counteracted with dsRNA transfection and suppressed the NCL expression; thus, emphasizing the dynamic attributes of the probe. These collective findings highlight the robust versatility of our aptamer as a powerful tool for imaging cell surfaces, holding promising implications for cancer cell identification and the detection of RSV infections.

Lipopolysaccharide Activating NF-kB Signaling by Regulates HTRA1 Expression in Human Retinal Pigment Epithelial Cells.

Inflammation and elevated expression of high temperature requirement A serine peptidase 1 (HTRA1) are known high risk factors for age-related macular degeneration (AMD). However, the specific mechanism that HTRA1 causes AMD and the relationship between HTRA1 and inflammation remains unclear. We found that lipopolysaccharide (LPS) induced inflammation enhanced the expression of HTRA1, NF-κB, and p-p65 in ARPE-19 cells. Overexpression of HTRA1 up-regulated NF-κB expression, and on the other hand knockdown of HTRA1 down-regulated the expression of NF-κB. Moreover, NF-κB siRNA has no significant effect on the expression of HTRA1, suggesting HTRA1 works upstream of NF-κB. These results demonstrated that HTRA1 plays a pivotal role in inflammation, explaining possible mechanism of overexpressed HTRA1-induced AMD. Celastrol, a very common anti-inflammatory and antioxidant drug, was found to suppress inflammation by inhibiting phosphorylation of p65 protein efficaciously in RPE cells, which may be applied to the therapy of age-related macular degeneration.

A Semisynthetic Bioluminescence Sensor for Ratiometric Imaging of Metal Ions In Vivo Using DNAzymes Conjugated to An Engineered Nano-Luciferase.

DNA-based probes have gained significant attention as versatile tools for biochemical analysis, benefiting from their programmability and biocompatibility. However, most existing DNA-based probes rely on fluorescence as the signal output, which can be problematic due to issues like autofluorescence and scattering when applied in complex biological materials such as living cells or tissues. Herein, we report the development of bioluminescent nucleic acid (bioLUNA) sensors that offer laser excitation-independent and ratiometric imaging of the target in vivo. The system is based on computational modelling and mutagenesis investigations of a genetic fusion between circular permutated Nano-luciferase (NLuc) and HaloTag, enabling the conjugation of the protein with a DNAzyme. In the presence of Zn2+ , the DNAzyme sensor releases the fluorophore-labelled strand, leading to a reduction in bioluminescent resonance energy transfer (BRET) between the luciferase and fluorophore. Consequently, this process induces ratiometric changes in the bioluminescent signal. We demonstrated that this bioLUNA sensor enabled imaging of both exogenous Zn2+ in vivo and endogenous Zn2+ efflux in normal epithelial prostate and prostate tumors. This work expands the DNAzyme sensors to using bioluminescence and thus has enriched the toolbox of nucleic acid sensors for a broad range of biomedical applications.

Noninvasive and Spatiotemporal Control of DNAzyme-Based Imaging of Metal Ions In Vivo Using High-Intensity Focused Ultrasound.

Detecting metal ions in vivo with a high spatiotemporal resolution is critical to understanding the roles of the metal ions in both healthy and disease states. Although spatiotemporal controls of metal-ion sensors using light have been demonstrated, the lack of penetration depth in tissue and in vivo has limited their application. To overcome this limitation, we herein report the use of high-intensity focused ultrasound (HIFU) to remotely deliver on-demand, spatiotemporally resolved thermal energy to activate the DNAzyme sensors at the targeted region both in vitro and in vivo. A Zn2+-selective DNAzyme probe is inactivated by a protector strand to block the formation of catalytic enzyme structure, which can then be activated by an HIFU-induced increase in the local temperature. With this design, Zn2+-specific fluorescent resonance energy transfer (FRET) imaging has been demonstrated by the new DNAzyme-HIFU probes in both HeLa cells and mice. The current method can be applied to monitor many other metal ions for in vivo imaging and medical diagnosis using metal-specific DNAzymes that have either been obtained or can be selected using in vitro selection.

Genetic landscape of FOXC2 mutations in lymphedema-distichiasis syndrome: Different mechanism of pathogenicity for mutations in different domains.

Lymphedema-dissociated syndrome (LDS), of which the pathogenesis is not fully understood, afflicts many patients. In this study, we investigated the effect of FOXC2 gene loss-of-function on the development of LDS disease. Two Han Chinese families with LDS were recruited in this study, pathogenic mutations were identified by Sanger sequencing. Reverse-transcription PCR, subcellular localization, dual fluorescein enzymes, and other in vitro experiments were used to study the functional effects of eight FOXC2 mutations. Two pathogenic FOXC2 duplication mutations (c.930_936dup and c.931-937dup) were identified in the two families. Both mutations caused uneven distribution in the nucleus and a chromatin contraction phenotype, weakening the DNA binding activity and transcription activity. We then performed functional analysis on six additional mutations in different domains of FOXC2 that were reported to cause LDS. We found mutations located in the forkhead domain and central region dramatically reduced the transactivation ability, while mutations in activation domain-2 enhanced this ability. All 8 mutations down-regulated the transcription of ANGPT2 and affected the activity of the ERK-RAS pathway, which may cause abnormal formation of lymphatic vessels. Our findings also showed that all 8 mutations decreased the ability of interaction between FOXC2 and the Wnt4 promoter, suggesting mutations in FOXC2 may also affect the Wnt4-Frizzled-RYK signaling pathway, leading the abnormal differentiation of the meibomian glands into hair follicle cells during the embryonic period and causing distichiasis. This study expanded and revealed the potential pathogenesis mechanism.

Probing the Functional and Structural Connectivity Underlying EEG Traveling Waves.

Neural oscillations play an important role in the maintenance of brain function by regulating multi-scale neural activity. Characterizing the traveling properties of EEG is helpful for understanding the spatiotemporal dynamics of neural oscillations. However, traveling EEG based on non-invasive approach has little been investigated, and the relationship with brain intrinsic connectivity is not well known. In this study, traveling EEG of different frequency bands on the scalp in terms of the center of mass (EEG-CM) was examined. Then, two quantitative indexes describing the spatiotemporal features of EEG-CM were proposed, i.e., the traveling lateralization and velocity of EEG-CM. Further, based on simultaneous EEG-MRI approach, the relationship between traveling EEG-CM and the resting-state functional networks, as well as the microstructural connectivity of white matter was investigated. The results showed that there was similar spatial distribution of EEG-CM under different frequency bands, while the velocity of rhythmic EEG-CM increased in higher frequency bands. The lateralization of EEG-CM in low frequency bands (< 30 Hz) demonstrated negative relationship with the basal ganglia network (BGN). In addition, the velocity of the traveling EEG-CM was associated with the fractional anisotropy (FA) in corpus callosum and corona radiate. These results provided valid quantitative EEG index for understanding the spatiotemporal characteristics of the scalp EEG, and implied that the EEG dynamics were representations of functional and structural organization of cortical and subcortical structures.

Genetic factors define CPO and CLO subtypes of nonsyndromicorofacial cleft.

Nonsyndromic orofacial cleft (NSOFC) is a severe birth defect that occurs early in embryonic development and includes the subtypes cleft palate only (CPO), cleft lip only (CLO) and cleft lip with cleft palate (CLP). Given a lack of specific genetic factor analysis for CPO and CLO, the present study aimed to dissect the landscape of genetic factors underlying the pathogenesis of these two subtypes using 6,986 cases and 10,165 controls. By combining a genome-wide association study (GWAS) for specific subtypes of CPO and CLO, as well as functional gene network and ontology pathway analysis, we identified 18 genes/loci that surpassed genome-wide significance (P < 5 × 10-8) responsible for NSOFC, including nine for CPO, seven for CLO, two for both conditions and four that contribute to the CLP subtype. Among these 18 genes/loci, 14 are novel and identified in this study and 12 contain developmental transcription factors (TFs), suggesting that TFs are the key factors for the pathogenesis of NSOFC subtypes. Interestingly, we observed an opposite effect of the genetic variants in the IRF6 gene for CPO and CLO. Moreover, the gene expression dosage effect of IRF6 with two different alleles at the same single-nucleotide polymorphism (SNP) plays important roles in driving CPO or CLO. In addition, PAX9 is a key TF for CPO. Our findings define subtypes of NSOFC using genetic factors and their functional ontologies and provide a clue to improve their diagnosis and treatment in the future.

Heterozygote loss-of-function variants in the LRP5 gene cause familial exudative vitreoretinopathy.

BACKGROUND: Familial exudative vitreoretinopathy (FEVR) is an inherited ocular disease with clinical manifestations of aberrant retinal vasculature. We aimed to identify novel causative variants responsible for FEVR and provided evidence for the genetic counselling of FEVR. METHODS: We applied whole-exome sequencing (WES) on the genomic DNA samples from the probands and performed Sanger sequencing for variant validation. Western blot analysis and luciferase assays were performed to test the expression levels and the activity of mutant proteins. RESULTS: We identified one novel heterozygous nonsense variant, and three novel heterozygous frameshift variants including c.1801G>T (p.G601*), c.1965delC (p.H656Tfs*41), c.4445delC (p.S1482Cfs*17), and c.4482delC (p.P1495Rfs*4), which disabled the function of LRP5 on the Norrin/ß-catenin signalling. Overexpression of variant-carrying LRP5 proteins resulted in down regulation of the protein levels of ß-catenin and the Norrin/ß-catenin signalling target genes c-Myc and Glut1. CONCLUSION: Our study showed that four inherited LRP5 variants can cause autosomal dominant FEVR via down regulation of Norrin/ß-catenin signalling and expanded the spectrum of FEVR-associated LRP5 variants.

Sequencing and Genomic Analysis of Sorghum DNA Introgression Variant Line R21 and Recipient Rice Jin Hui 1 Revealed Repetitive Element Variation.

Transferring the genome of distant species to crops is an efficient way to create new germplasms. However, the molecular mechanisms involved are unclear. In this study, a new rice restorer line R21 with heat tolerance was created by introgressing the genomic DNA of sorghum into the recipient restorer line Jin Hui 1. Assembly of rice R21 and Jin Hui 1 genomes was performed using PacBio sequencing technology. Comparative genome analysis and coverage statistics showed that the repetitive sequence atr0026 was a candidate introgression fragment of sorghum DNA. Sequence similarity analysis revealed that atr0026 was distributed at different copy numbers on the telomeric position of chromosomes 9 or 10 in R21, Jin Hui 1, and several rice varieties, indicating that the repetitive sequence from sorghum was highly conserved in rice. The repeat annotation in Gramineae indicated that ribosomal DNA loci that existed in atr0026 may be cause a rearrangement of chromosomes 9 and 10 of the R21 genome, resulting in a copy number variation at the 5' end of it. Our study lays the foundation for further elucidation of the molecular mechanisms underlying the heat tolerance of sorghum DNA introgression variant line R21, which is of great significance for guiding crop genetic breeding.

[Association analysis of seven single nucleotide polymorphisms identified by genome-wide association study with age-related macular degeneration among ethnic Han Chinese population].

OBJECTIVE: To assess the association of 7 single nucleotide polymorphisms (SNPs) including rs13278062 (TNFRSF10A), rs3750846 (ARMS2-HTRA1), rs429358 (APOE), rs5817082 (CEPT), rs2043085 (LIPC), rs1626340 (TGFBR1), and rs8135665 (SLC16A8) identified through genome-wide association study (GWAS) with age-related macular degeneration (AMD) among ethnic Han Chinese from Sichuan, China. METHODS: A cohort of 576 AMD patients and 572 healthy controls were enrolled in a case-control study. The SNPs were genotyped by a Mass array MALDI-TOF System. On the premise that the genotype distribution of each SNP locus in both groups satisfied Hardy-Weinberg equilibrium, the genetic pattern was analyzed and the scores of allele and genotype frequencies ware compared. RESULTS: There was a significant association between TNFRSF10A rs13278062 and AMD under the heterozygous model (P = 0.000, OR = 1.529, 95%CI = 1.196-1.954) and the dominant model (P = 0.002, OR = 1.459, 95%CI = 1.154-1.865), suggesting that subjects carrying rs13278062GT and rs13278062TT + GT are more likely to develop the AMD, whereas no significant difference was observed for rs13278062 under other models. No association was detected with the other six SNPs and AMD under various genetic models. CONCLUSION: This case-control association study has indicated that TNFRSF10A rs13278062 is associated with AMD under the heterozygous and dominant models, suggesting that the TNFRSF10A variant may be involved in the development of AMD among ethnic Han Chinese population.

Combination Cancer Treatment: Using Engineered DNAzyme Molecular Machines for Dynamic Inter- and Intracellular Regulation.

Despite the promise of combination cancer therapy, it remains challenging to develop targeted strategies that are nontoxic to normal cells. Here we report a combination therapeutic strategy based on engineered DNAzyme molecular machines that can promote cancer apoptosis via dynamic inter- and intracellular regulation. To achieve external regulation of T-cell/cancer cell interactions, we designed a DNAzyme-based molecular machine with an aptamer and an i-motif, as the MUC-1-selective aptamer allows the specific recognition of cancer cells. The i-motif is folded under the tumor acidic microenvironment, shortening the intercellular distance. As a result, T-cells are released by metal ion activated DNAzyme cleavage. To achieve internal regulation of mitochondria, we delivered another DNAzyme-based molecular machine with mitochondria-targeted peptides into cancer cells to induce mitochondria aggregation. Our strategy achieved an enhanced killing effect in zinc deficient cancer cells.

PNA-Assisted DNAzymes to Cleave Double-Stranded DNA for Genetic Engineering with High Sequence Fidelity.

DNAzymes have been widely used in many sensing and imaging applications but have rarely been used for genetic engineering since their discovery in 1994, because their substrate scope is mostly limited to single-stranded DNA or RNA, whereas genetic information is stored mostly in double-stranded DNA (dsDNA). To overcome this major limitation, we herein report peptide nucleic acid (PNA)-assisted double-stranded DNA nicking by DNAzymes (PANDA) as the first example to expand DNAzyme activity toward dsDNA. We show that PANDA is programmable in efficiently nicking or causing double strand breaks on target dsDNA, which mimics protein nucleases and can act as restriction enzymes in molecular cloning. In addition to being much smaller than protein enzymes, PANDA has a higher sequence fidelity compared with CRISPR/Cas under the condition we tested, demonstrating its potential as a novel alternative tool for genetic engineering and other biochemical applications.