Multiplexed sequential imaging in living cells with orthogonal fluorogenic RNA aptamer/dye pairs.

Detecting multiple targets in living cells is important in cell biology. However, multiplexed fluorescence imaging beyond two-to-three targets remains a technical challenge. Herein, we introduce a multiplexed imaging strategy, 'sequential Fluorogenic RNA Imaging-Enabled Sensor' (seqFRIES), which enables live-cell target detection via sequential rounds of imaging-and-stripping. In seqFRIES, multiple orthogonal fluorogenic RNA aptamers are genetically encoded inside cells, and then the corresponding cell membrane permeable dye molecules are added, imaged, and rapidly removed in consecutive detection cycles. As a proof-of-concept, we have identified in this study four fluorogenic RNA aptamer/dye pairs that can be used for highly orthogonal and multiplexed imaging in living bacterial and mammalian cells. After further optimizing the cellular fluorescence activation and deactivation kinetics of these RNA/dye pairs, the whole four-color semi-quantitative seqFRIES process can be completed in ∼20 min. Meanwhile, seqFRIES-mediated simultaneous detection of critical signalling molecules and mRNA targets was also achieved within individual living cells. We expect our validation of this new seqFRIES concept here will facilitate the further development and potential broad usage of these orthogonal fluorogenic RNA/dye pairs for multiplexed and dynamic live-cell imaging and cell biology studies.

Targeted RNA condensation in living cells via genetically encodable triplet repeat tags.

Living systems contain various membraneless organelles that segregate proteins and RNAs via liquid-liquid phase separation. Inspired by nature, many protein-based synthetic compartments have been engineered in vitro and in living cells. Here, we introduce a genetically encoded CAG-repeat RNA tag to reprogram cellular condensate formation and recruit various non-phase-transition RNAs for cellular modulation. With the help of fluorogenic RNA aptamers, we have systematically studied the formation dynamics, spatial distributions, sizes and densities of these cellular RNA condensates. The cis- and trans-regulation functions of these CAG-repeat tags in cellular RNA localization, life time, RNA-protein interactions and gene expression have also been investigated. Considering the importance of RNA condensation in health and disease, we expect that these genetically encodable modular and self-assembled tags can be widely used for chemical biology and synthetic biology studies.

Chemical composition determination and transcriptomic analyses provide insight into the differences between wild and grafted Semen Ziziphi Spinosae.

Semen Ziziphi Spinosae (SZS) is a traditional Chinese herbal medicine widely used to treat insomnia and anxiety in clinical practice. Currently, the demand for SZS is increasing every year, but the production of wild SZS is unstable due to environmental factors. Grafting sour jujube scions onto sour jujube or jujube tree stocks can achieve a high production rate within a short period of time. However, the effects of grafting on the quality of SZS have not been reported. This study investigated the differences between wild-type and grafted SZS from three aspects: phenotype, chemical composition, and molecular mechanism. The findings revealed that the grafted specimens were generally larger in morphology and lighter in color than the wild-type samples. The dimensions of both the grafted specimens were generally larger than those of the wild specimens. The HPLC-ELSD results revealed that the three main chemical components in the grafted SZS, namely, spinosin, jujuboside A, and jujuboside B, had higher contents than their wild-type counterparts. Comprehensive transcriptome sequencing analysis and KEGG annotation revealed that DEG enrichment between grafted and wild-type SZS occurred mainly during stress resistance and rootstock scion healing. There were 23 DEGs that may encode enzymes involved in the biosynthetic pathway of flavonoids and 21 genes encoding terpenoid saponins. Further investigation revealed that the expression of the genes C4H, CHS, CHI, and F3'5'H in the flavonoid biosynthesis pat.hway and HMGR, MVK, MVD, and FPPS in the saponin biosynthesis pathway accounted for the difference in quality between grafted and wild SZS. Furthermore, WGCNA identified 15 core genes related to medicinal ingredients between grafted and wild SZS. These results provide support for further research on the differences in the quality of medicinal ingredients between grafted and wild SZS.

Central nervous system anomalies in 41 Chinese children incontinentia pigmenti.

INTRODUCTION: Incontinentia pigmenti (IP) is a rare neuroectodermal dysplasia caused by a defect in the IKBKG gene. The pathogenesis of central nervous system injury is believed to be related to microvascular ischemia. Currently, few treatment strategies are available for the inflammatory phase. MATERIALS AND METHODS: This retrospective descriptive analysis included the clinical data of 41 children with IP collected from 2007 to 2021 in Xi'an, China, comprising clinical characteristics, imaging findings, blood cell analysis, skin histopathology, and genetic data. RESULTS: Fourteen children (34%) aged 4 days to 5 months exhibited clinical signs and symptoms, including convulsions, delayed psychomotor development following neurological damage, and revealed significant MRI abnormalities, including ischemia, hypoxia, cerebral hypoperfusion, hemorrhage, encephalomalacia, and cerebral atrophy. Eight of the 24 patients (33%) presented with retinal vascular tortuosity and telangiectasis, accompanied by neovascularization and hemorrhage. Thirty-eight children (93%) had elevated eosinophils (mean: 3.63 ± 4.46 × 109), and 28 children (68%) had significantly elevated platelets (mean: 420.16 ± 179.43 × 109). Histopathology of skin revealed microvascular extravasation and vasodilation with perivascular and intravascular eosinophilic infiltration. CONCLUSION: Brain injury in IP occurs during infancy until 5 months of age, which is also the acute dermatitis phase accompanied by eosinophilia and an increased platelet count. This study provides evidence of microvascular damage to the skin and fundus during the inflammatory phase. The mechanism of microvascular damage may be similar to that in the brain.

Intestinal metabolite xylulose inhibits colorectal cancer by inducing apoptosis through the MAPK signalling pathway.

BACKGROUND: The intestinal metabolites are involved in the initiation, progression and metastasis of colorectal cancer (CRC). They are a potential source of agents for cancer therapy. Our previous study identified altered faecal metabolites between CRC patients and healthy volunteers. However, no specific metabolite was clearly illustrated for CRC therapy. RESULTS: We found that the level of xylulose was lower in the stools of CRC patients than in those of healthy volunteers. Xylulose inhibited cell growth without affecting the cell cycle by inducing apoptosis in CRC cells, which was evidenced by increased expression of the proapoptotic proteins C-PARP and C-Caspase3 and decreased expression of the antiapoptotic protein BCL-2 in CRC cells. Mechanistically, xylulose reduced the activity of the MAPK signalling pathway, represented by reduced phosphorylation of JNK, ERK, and P38. Furthermore, an ALI model was used to show the tumour killing ability of xylulose on human CRC spheres, as well as human colorectal adenoma (AD) spheres. CONCLUSION: Xylulose inhibits CRC growth by inducing apoptosis through attenuation of the MAPK signalling pathway. These results suggest that xylulose may serve as an effective agent for CRC therapy.

Improving spatial resolution and diagnostic confidence with thinner slice and deep learning image reconstruction in contrast-enhanced abdominal CT.

OBJECTIVE: To evaluate image quality and diagnostic confidence improvement using a thin slice and a deep learning image reconstruction (DLIR) in contrast-enhanced abdominal CT. METHODS: Forty patients with hepatic lesions in enhanced abdominal CT were retrospectively analyzed. Images in the portal phase were reconstructed at 5 mm and 1.25 mm slice thickness using the 50% adaptive statistical iterative reconstruction (ASIR-V) (ASIR-V50%) and at 1.25 mm using DLIR at medium (DLIR-M) and high (DLIR-H) settings. CT number and standard deviation of the hepatic parenchyma, spleen, portal vein, and subcutaneous fat were measured, and contrast-to-noise ratio (CNR) was calculated. Edge-rise-slope (ERS) was measured on the portal vein to reflect spatial resolution and the CT number skewness on liver parenchyma was calculated to reflect image texture. Two radiologists blindly assessed the overall image quality including subjective noise, image contrast, visibility of small structures using a 5-point scale, and object sharpness and lesion contour using a 4-point scale. RESULTS: For the 1.25-mm images, DLIR significantly reduced image noise, improved CNR and overall subjective image quality compared to ASIR-V50%. Compared to the 5-mm ASIR-V50% images, DLIR images had significantly higher scores in the visibility and contour for small structures and lesions; as well as significantly higher ERS and lower CT number skewness. At a quarter of the signal strength, the 1.25-mm DLIR-H images had a similar subjective noise score as the 5-mm ASIR-V50% images. CONCLUSION: DLIR significantly reduces image noise and maintains a more natural image texture; image spatial resolution and diagnostic confidence can be improved using thin slice images and DLIR in abdominal CT. KEY POINTS: • DLIR further reduces image noise compared with ASIR-V while maintaining favorable image texture. • In abdominal CT, thinner slice images improve image spatial resolution and small object visualization but suffer from higher image noise. • Thinner slice images combined with DLIR in abdominal CT significantly suppress image noise for detecting low-density lesions while significantly improving image spatial resolution and overall image quality.

Advanced DNA Zipper Probes for Detecting Cell Membrane Lipid Domains.

The cell membrane is a complex mixture of lipids, proteins, and other components. By forming dynamic lipid domains, different membrane molecules can selectively interact with each other to control cell signaling. Herein, we report several new types of lipid-DNA conjugates, termed as "DNA zippers", which can be used to measure cell membrane dynamic interactions and the formation of lipid domains. Dependent on the choice of lipid moieties, cholesterol- and sphingomyelin-conjugated DNA zippers specifically locate in and detect membrane lipid-ordered domains, while in contrast, a tocopherol-DNA zipper can be applied for the selective imaging of lipid-disordered phases. These versatile and programmable probes can be further engineered into membrane competition assays to simultaneously detect multiple types of membrane dynamic interactions. These DNA zipper probes can be broadly used to study the correlation between lipid domains and various cellular processes, such as the epithelial-mesenchymal transition.

Genome-Wide Characterization of B-Box Gene Family in Salvia miltiorrhiza.

B-box (BBX) is a type of zinc finger transcription factor that contains a B-box domain. BBX transcription factors play important roles in plant photomorphogenesis, signal transduction, as well as abiotic and biological stress responses. However, the BBX gene family of Salvia miltiorrhiza has not been systematically investigated to date. For this study, based on the genomic data of Salvia miltiorrhiza, 27 SmBBXs genes were identified and clustered into five evolutionary branches according to phylogenetic analysis. The promoter analysis suggested that SmBBXs may be involved in the regulation of the light responses, hormones, stress signals, and tissue-specific development. Based on the transcriptome data, the expression patterns of SmBBXs under different abiotic stresses and plant hormones were analyzed. The results revealed that the expressions of the SmBBXs genes varied under different conditions and may play essential roles in growth and development. The transient expression analysis implied that SmBBX1, SmBBX4, SmBBX9, SmBBX20, and SmBBX27 were in the nucleus. A transcriptional activation assay showed SmBBX1, SmBBX4, SmBBX20, and SmBBX24 had transactivation activities, while SmBBX27 had none. These results provided a basis for further research on the role of SmBBXs in the development of Salvia miltiorrhiza.

Development of one-step multiplex RT-PCR assay for rapid simultaneous detection of five RNA viruses and Acidovorax citrulli in major cucurbitaceous crops in China.

Cucurbitaceous fruits and vegetables are important crops. Viral and bacterial diseases cause substantial economic losses to cucurbit crops globally. For rapid detection of these pathogens and improved disease control, a one-step multiplex reverse-transcription polymerase chain reaction (mRT-PCR) system was created. This method allowed for the concurrent detection of Tobacco mosaic virus (TMV), Zucchini yellow mosaic virus (ZYMV), Watermelon mosaic virus (WMV), Cucumber green mottle mosaic virus (CGMMV), Cucumber mosaic virus (CMV), and Acidovorax citrulli. Five pairs of specific primers were created according to the conserved regions around the coat protein (CP) genes of each virus, and one pair was based on the A. citrulli internal transcribed spacer (ITS). To limit false negatives, one pair of primers, created based on the Transcriptional elongation factor 1-α (EF1-α) from the major cucurbitaceous crop species, was put into the mRT-PCR reaction system. Primer concentrations, annealing temperature, extension time, and amplification cycles were optimized. Anticipated fragments of 152 bp (TMV), 205 bp (ZYMV), 318 bp (WMV), 419 bp (CGMMV), 529 bp (CMV), 662 bp (A. citrulli), and 821 bp (EF1-α) were amplified by the multiplex RT-PCR system, and their origin was established via DNA sequencing. This method was successfully used to examine field-collected seed samples of cucurbitaceous crops from China. The results demonstrated that the one-step mRT-PCR technique is a quick, efficient, and sensitive assay for the concurrent detection of six pathogens of cucurbits. It provides a method for monitoring and preventing these diseases.

Evaluation of Droplet Digital PCR for the Detection of Black Canker Disease in Tomato.

Clavibacter michiganensis subsp. michiganensis, the cause of bacterial canker disease, is one of the most destructive pathogens in greenhouse and field tomato. The pathogen is now present in all main production areas of tomato and is widely distributed in the European and Mediterranean Plant Protection Organization region. The inspection and quarantine of the plant pathogens relies heavily on accurate detection tools. Primers and probes reported in previous studies do not distinguish the C. michiganensis subsp. michiganensis pathogen from other closely related subspecies of C. michiganensis, especially the nonpathogenic subspecies that were identified from tomato seeds recently. Here, we have developed a droplet digital PCR (ddPCR) method for the identification of this specific bacterium with primers/TaqMan probe set designed based on the pat-1 gene of C. michiganensis subsp. michiganensis. This new primers/probe set has been evaluated by real-time PCR (qPCR) and ddPCR. The detection results suggest that the ddPCR method established in this study was highly specific for the target strains. The result showed the positive amplification for all five C. michiganensis subsp. michiganensis strains, and no amplification was observed for the other 43 tested bacteria, including the closely related C. michiganensis strains. The detection threshold of ddPCR was 10.8 CFU/ml for both pure C. michiganensis subsp. michiganensis cell suspensions and infected tomato seed, which was 100-fold more sensitive than qPCR performed using the same primers and probe. The data obtained suggest that our established ddPCR could detect C. michiganensis subsp. michiganensis even with low bacterial load, which could facilitate both C. michiganensis subsp. michiganensis inspection for pathogen quarantine and the routine pathogen detection for disease control of black canker in tomato.

Image quality improvement in low-dose chest CT with deep learning image reconstruction.

OBJECTIVES: To investigate the clinical utility of deep learning image reconstruction (DLIR) for improving image quality in low-dose chest CT in comparison with 40% adaptive statistical iterative reconstruction-Veo (ASiR-V40%) algorithm. METHODS: This retrospective study included 86 patients who underwent low-dose CT for lung cancer screening. Images were reconstructed with ASiR-V40% and DLIR at low (DLIR-L), medium (DLIR-M), and high (DLIR-H) levels. CT value and standard deviation of lung tissue, erector spinae muscles, aorta, and fat were measured and compared across the four reconstructions. Subjective image quality was evaluated by two blind readers from three aspects: image noise, artifact, and visualization of small structures. RESULTS: The effective dose was 1.03 ± 0.36 mSv. There was no significant difference in CT values of erector spinae muscles and aorta, whereas the maximum difference for lung tissue and fat was less than 5 HU among the four reconstructions. Compared with ASiR-V40%, the DLIR-L, DLIR-M, and DLIR-H reconstructions reduced the noise in aorta by 11.44%, 33.03%, and 56.1%, respectively, and had significantly higher subjective quality scores in image artifacts (all p < 0.001). ASiR-V40%, DLIR-L, and DLIR-M had equivalent score in visualizing small structures (all p > 0.05), whereas DLIR-H had slightly lower score. CONCLUSIONS: Compared with ASiR-V40%, DLIR significantly reduces image noise in low-dose chest CT. DLIR strength is important and should be adjusted for different diagnostic needs in clinical application.

Incidence of extrauterine growth retardation and its risk factors in very preterm infants during hospitalization: a multicenter prospective study. / ä½é™¢æžæ—©äº§å„¿å®«å¤–ç”Ÿé•¿è¿Ÿç¼“åŠç›¸å ³å› ç´ çš„å¤šä¸­å¿ƒå‰çž»æ€§ç ”ç©¶.

OBJECTIVES: To investigate the incidence of extrauterine growth retardation (EUGR) and its risk factors in very preterm infants (VPIs) during hospitalization in China. METHODS: A prospective multicenter study was performed on the medical data of 2 514 VPIs who were hospitalized in the department of neonatology in 28 hospitals from 7 areas of China between September 2019 and December 2020. According to the presence or absence of EUGR based on the evaluation of body weight at the corrected gestational age of 36 weeks or at discharge, the VPIs were classified to two groups: EUGR group (n=1 189) and non-EUGR (n=1 325). The clinical features were compared between the two groups, and the incidence of EUGR and risk factors for EUGR were examined. RESULTS: The incidence of EUGR was 47.30% (1 189/2 514) evaluated by weight. The multivariate logistic regression analysis showed that higher weight growth velocity after regaining birth weight and higher cumulative calorie intake during the first week of hospitalization were protective factors against EUGR (P<0.05), while small-for-gestational-age birth, prolonged time to the initiation of total enteral feeding, prolonged cumulative fasting time, lower breast milk intake before starting human milk fortifiers, prolonged time to the initiation of full fortified feeding, and moderate-to-severe bronchopulmonary dysplasia were risk factors for EUGR (P<0.05). CONCLUSIONS: It is crucial to reduce the incidence of EUGR by achieving total enteral feeding as early as possible, strengthening breastfeeding, increasing calorie intake in the first week after birth, improving the velocity of weight gain, and preventing moderate-severe bronchopulmonary dysplasia in VPIs.

Description of Clavibacter zhangzhiyongii sp. nov., a phytopathogenic actinobacterium isolated from barley seeds, causing leaf brown spot and decline.

Clavibacter michiganensis is a Gram-stain-positive bacterium with eight subspecies, five of which have been redefined as different species on the basis of their genome sequence data. On the basis of the results of phylogenetic analysis of dnaA gene sequences, strains of members of the genus Clavibacter isolated from barley have been grouped in a separate clade from other species and subspecies of the genus Clavibacter. In this study, the biochemical, physiological, fatty acids and genetic characteristics of strains DM1T and DM3, which represented the barley isolates, were examined. On the basis of results from multi-locus sequence typing and other biochemical and physiological features, including colony colour, carbon source utilisation and enzyme activities, DM1T and DM3 are categorically differentiated from the aforementioned eight species and subspecies of the genus Clavibacter. Moreover, the results of genomic analysis reveal that the DNA G+C contents of DM1T and DM3 are 73.7 and 73.5 %, respectively, and the average nucleotide identity (ANI) values between DM1T and DM3 and other species and subspecies range from 90.4 to 92.0 %. The ANI value between DM1T and DM3 is 98.0 %. These results indicate that DM1T and DM3 are distinct from other known species and subspecies of the genus Clavibacter. Therefore, we propose a novel species, C. zhangzhiyongii, with DM1T (=CFCC 16553 T=LMG 31970T) as the type strain.

Species differences in the CYP3A-catalyzed metabolism of TPN729, a novel PDE5 inhibitor.

TPN729 is a novel phosphodiesterase 5 (PDE5) inhibitor used to treat erectile dysfunction in men. Our previous study shows that the plasma exposure of metabolite M3 (N-dealkylation of TPN729) in humans is much higher than that of TPN729. In this study, we compared its metabolism and pharmacokinetics in different species and explored the contribution of its main metabolite M3 to pharmacological effect. We conducted a combinatory approach of ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry-based metabolite identification, and examined pharmacokinetic profiles in monkeys, dogs, and rats following TPN729 administration. A remarkable species difference was observed in the relative abundance of major metabolite M3: i.e., the plasma exposure of M3 was 7.6-fold higher than that of TPN729 in humans, and 3.5-, 1.2-, 1.1-fold in monkeys, dogs, and rats, respectively. We incubated liver S9 and liver microsomes with TPN729 and CYP3A inhibitors, and demonstrated that CYP3A was responsible for TPN729 metabolism and M3 formation in humans. The inhibitory activity of M3 on PDE5 was 0.78-fold that of TPN729 (The IC50 values of TPN729 and M3 for PDE5A were 6.17 ± 0.48 and 7.94 ± 0.07 nM, respectively.). The plasma protein binding rates of TPN729 and M3 in humans were 92.7% and 98.7%, respectively. It was astonishing that the catalyzing capability of CYP3A4 in M3 formation exhibited seven-fold disparity between different species. M3 was an active metabolite, and its pharmacological contribution was equal to that of TPN729 in humans. These findings provide new insights into the limitation and selection of animal model for predicting the clinical pharmacokinetics of drug candidates metabolized by CYP3A4.

In Situ Genetically Cascaded Amplification for Imaging RNA Subcellular Locations.

In situ amplification methods, such as hybridization chain reaction, are valuable tools for mapping the spatial distribution and subcellular location of target analytes. However, the live-cell applications of these methods are still limited due to challenges in the probe delivery, degradation, and cytotoxicity. Herein, we report a novel genetically encoded in situ amplification method to noninvasively image the subcellular location of RNA targets in living cells. In our system, a fluorogenic RNA reporter, Broccoli, was split into two nonfluorescent fragments and conjugated to the end of two RNA hairpin strands. The binding of one target RNA can then trigger a cascaded hybridization between these hairpin pairs and thus activate multiple Broccoli fluorescence signals. We have shown that such an in situ amplified strategy can be used for the sensitive detection and location imaging of various RNA targets in living bacterial and mammalian cells. This new design principle provides an effective and versatile platform for tracking various intracellular analytes.

Characterization and source identification of PM2.5-bound polycyclic aromatic hydrocarbons in urban, suburban, and rural ambient air, central China during summer harvest.

Characterization and source identification of PM2.5-bound polycyclic aromatic hydrocarbons (PAHs) are conducted in urban Wuhan (WH), suburban Pingdingshan (PDS), and rural Suizhou (SZ) in China during summer harvest. This study analyzes 16 priority PAHs with 38 PM.2.5 samples in June. PAHs had similar physical-chemical properties like polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs), which had been listed as Priority Pollutants. The concentration and detection frequency of OCPs and PCBs were considerably lower than those of PAHs in PM2.5. Results indicate that PDS adjoining the highway has the highest PM2.5-bound PAHs. SZ possesses the lowest concentration of PAHs. Principal component analysis and multivariate linear regression model and molecular diagnostic ratio distinguish the sources. Vehicle emissions and coal combustion are extracted in three sites, while the source of PDS also includes gas combustion. SZ was affected by gas combustion and petroleum. The potential source contribution function and the concentration-weighted trajectory track the potential pollution area. The sampling places might be affected by the local sources and short distance transmission cannot be neglected. The incremental lifetime cancer risks (ILCRs) model evaluates the exposure risk of PAHs. According to the ILCR model, WH and PDS are exposed to harmful PAHs. By contrast, SZ is a substantially safe place.

Fate of PM2.5-bound PAHs in Xiangyang, central China during 2018 Chinese spring festival: Influence of fireworks burning and air-mass transport.

Variations of levels, possible source and air mass transmission were investigated for 16 USEPA priority-controlled PAHs in PM2.5 during 2018 Chinese Spring Festival (CSF) in Xiangyang City, central China which is the North-South pollutant airmass transport channel of China. Totally 37 samples were collected. Mass concentrations of Σ16PAHs for the Pre-CSF day (Pre-CSFD), during the CSF day (CSFD) and after the CSF day (Af-CSFD) are 33.78 ± 17.68 ng/m3, 22.98 ± 6.49 ng/m3, and 8.99 ± 4.44 ng/m3, respectively. High resolution samples showed that Σ16PAHs are higher in the morning (06:00-11:00) or afternoon (11:30-16:30), than those in the evening (17:00-22:00) and at night (22:30-05:30), whereas the result is reversed during the CSFD. Fireworks burning can obviously increase the mass concentration of PAHs. Air mass trajectory indicated that Xiangyang is a sink area of pollutants for northwest and southeast, and the sources of the northeast and southwest. The air mass only can be transmitted out through northeast and southwest. It is effective for improvement of air quality in Wuhan and Hunan to control fireworks emission in Henan and local areas. Fireworks burning was an important source for PAHs during CSFD, biomass, coal combustion, and traffic emission were the main sources of PAHs for Pre-CSFD and Af-CSFD periods. The health risk on the CSFD was higher than the acceptable levels, especially during the intensive fireworks burning, the risk value far exceed 1.0 × 10-4, controlling burning fireworks is required.

A Genetically Encoded RNA Photosensitizer for Targeted Cell Regulation.

Genetically encoded RNA devices have emerged for various cellular applications in imaging and biosensing, but their functions as precise regulators in living systems are still limited. Inspired by protein photosensitizers, we propose here a genetically encoded RNA aptamer based photosensitizer (GRAP). Upon illumination, the RNA photosensitizer can controllably generate reactive oxygen species for targeted cell regulation. The GRAP system can be selectively activated by endogenous stimuli and light of different wavelengths. Compared with their protein analogues, GRAP is highly programmable and exhibits reduced off-target effects. These results indicate that GRAP enables efficient noninvasive target cell ablation with high temporal and spatial precision. This new RNA regulator system will be widely used for optogenetics, targeted cell ablation, subcellular manipulation, and imaging.

AZGP1 is androgen responsive and involved in AR-induced prostate cancer cell proliferation and metastasis.

Alpha-2-glycoprotein 1, zinc-binding (AZGP1), known as zinc-alpha-2-glycoprotein (ZAG), is a multifunctional secretory glycoprotein and relevant to cancer metastasis. Little is known regarding the underlying mechanisms of AZGP1 in prostate cancer (PCa). In the present study, we report that AZGP1 is an androgen-responsive gene, which is involved in AR-induced PCa cell proliferation and metastasis. In clinical specimens, the expression of AZGP1 in PCa tissues is markedly higher than that in adjacent normal tissues. In cultures, expression of AZGP1 is upregulated by the androgen-AR axis at both messenger RNA and protein levels. Furthermore, Chip-Seq assay identifies canonical androgen-responsive elements (AREs) at AZGP1 enhancer; and dual-luciferase reporter assays reveal that the AREs is highly responsive to androgen whereas mutations of the AREs abolish the reporter activity. In addition, AZGP1 promotes G1/S phase transition and cell cycle progress by increasing cyclin D1 levels in PCa cells. Functional studies demonstrate that knocking down endogenous AZGP1 expression in LNCaP and CWR22Rv1 cells largely weaken androgen/AR axis-induced cell migration and invasion. In vivo xenotransplantation tumor experiments also show that AZGP1 involves in androgen/AR axis-mediated PCa cell proliferation. Taken together, our study implicates for the first time that AZGP1 is an AR target gene and is involved in androgen/AR axis-mediated cell proliferation and metastasis in primary PCa.

Engineering of ATP-Powered Photosensitizer for Targeted Recycling Activatable Imaging of MicroRNA and Controllable Cascade Amplification Photodynamic Therapy.

Owing to the low abundance of microRNAs (miRNAs) in living tumor cells, the development of intracellular cancer-relevant miRNA stimuli-activatable photosensitizers (PSs) for accurate imaging and efficient photodynamic therapy (PDT) of tumors in vivo is extremely challenging. Herein, we engineered a tumor targeting and intracellular trace miRNA-activatable nanophotosensitizer Y-motif/FA@HyNP on the basis of an endogenous ATP-powered strand-displacement cascade amplification strategy, which was prepared by assembly of a quencher BHQ2-labeled Y-motif DNA structure (containing ATP-binding aptamer and target miRNA-binding complementary sequence) on the surface of folate (FA) and amine-functionalized hybrid micellar nanoparticles. We showed that the fluorescence emissions at both 555 and 627 nm were effectively inhibited due to BHQ2 in Y-motif/FA@HyNPs, leading to negligible PDT efficacy. Once Y-motif/FA@HyNPs were selectively internalized into tumor cells via FA-receptor-mediated endocytosis, the intracellular trace target miRNA initiated the dissociation of the BHQ2-terminated sequences from Y-motif/FA@HyNPs by means of abundant endogenous ATP-powered strand-displacement reactions, causing remarkable fluorescence enhancement and cascade amplification PDT. The activated dual-color fluorescence emissions at 555 and 627 nm were feasible to achieve real-time, highly sensitive, and specific imaging of trace target miRNA in living tumor cells. With the guidance of excellent imaging in living mice, Y-motif/FA@HyNPs exhibited the precise and efficient PDT of tumors as well as insignificant side effects in vivo. This work revealed the great potential of using an integration of receptor-mediated cell uptake and target-triggered recycling cascade amplification strategy to design early cancer-relevant stimuli-activatable PSs for both fluorescence imaging and PDT ablation of tumors in vivo, which could effectively facilitate the timeliness and precision of early cancer diagnosis and therapy.