A vitamin-C-derived DNA modification catalysed by an algal TET homologue.

Methylation of cytosine to 5-methylcytosine (5mC) is a prevalent DNA modification found in many organisms. Sequential oxidation of 5mC by ten-eleven translocation (TET) dioxygenases results in a cascade of additional epigenetic marks and promotes demethylation of DNA in mammals1,2. However, the enzymatic activity and function of TET homologues in other eukaryotes remains largely unexplored. Here we show that the green alga Chlamydomonas reinhardtii contains a 5mC-modifying enzyme (CMD1) that is a TET homologue and catalyses the conjugation of a glyceryl moiety to the methyl group of 5mC through a carbon-carbon bond, resulting in two stereoisomeric nucleobase products. The catalytic activity of CMD1 requires Fe(II) and the integrity of its binding motif His-X-Asp, which is conserved in Fe-dependent dioxygenases3. However, unlike previously described TET enzymes, which use 2-oxoglutarate as a co-substrate4, CMD1 uses L-ascorbic acid (vitamin C) as an essential co-substrate. Vitamin C donates the glyceryl moiety to 5mC with concurrent formation of glyoxylic acid and CO2. The vitamin-C-derived DNA modification is present in the genome of wild-type C. reinhardtii but at a substantially lower level in a CMD1 mutant strain. The fitness of CMD1 mutant cells during exposure to high light levels is reduced. LHCSR3, a gene that is critical for the protection of C. reinhardtii from photo-oxidative damage under high light conditions, is hypermethylated and downregulated in CMD1 mutant cells compared to wild-type cells, causing a reduced capacity for photoprotective non-photochemical quenching. Our study thus identifies a eukaryotic DNA base modification that is catalysed by a divergent TET homologue and unexpectedly derived from vitamin C, and describes its role as a potential epigenetic mark that may counteract DNA methylation in the regulation of photosynthesis.

Efficient methods for multiple types of precise gene-editing in Chlamydomonas.

Precise gene-editing using CRISPR/Cas9 technology remains a long-standing challenge, especially for genes with low expression and no selectable phenotypes in Chlamydomonas reinhardtii, a classic model for photosynthesis and cilia research. Here, we developed a multi-type and precise genetic manipulation method in which a DNA break was generated by Cas9 nuclease and the repair was mediated using a homologous DNA template. The efficacy of this method was demonstrated for several types of gene editing, including inactivation of two low-expression genes (CrTET1 and CrKU80), the introduction of a FLAG-HA epitope tag into VIPP1, IFT46, CrTET1 and CrKU80 genes, and placing a YFP tag into VIPP1 and IFT46 for live-cell imaging. We also successfully performed a single amino acid substitution for the FLA3, FLA10 and FTSY genes, and documented the attainment of the anticipated phenotypes. Lastly, we demonstrated that precise fragment deletion from the 3'-UTR of MAA7 and VIPP1 resulted in a stable knock-down effect. Overall, our study has established efficient methods for multiple types of precise gene editing in Chlamydomonas, enabling substitution, insertion and deletion at the base resolution, thus improving the potential of this alga in both basic research and industrial applications.

Structural and functional regulation of Chlamydomonas lysosome-related organelles during environmental changes.

Lysosome-related organelles (LROs) are a class of heterogeneous organelles conserved in eukaryotes that primarily play a role in storage and secretion. An important function of LROs is to mediate metal homeostasis. Chlamydomonas reinhardtii is a model organism for studying metal ion metabolism; however, structural and functional analyses of LROs in C. reinhardtii are insufficient. Here, we optimized a method for purifying these organelles from 2 populations of cells: stationary phase or overloaded with iron. The morphology, elemental content, and lysosomal activities differed between the 2 preparations, even though both have phosphorus and metal ion storage functions. LROs in stationary phase cells had multiple non-membrane-bound polyphosphate granules to store phosphorus. Those in iron-overloaded cells were similar to acidocalcisomes (ACs), which have a boundary membrane and contain 1 or 2 large polyphosphate granules to store more phosphorus. We established a method for quantifying the capacity of LROs to sequester individual trace metals. Based on a comparative proteomic analysis of these 2 types of LROs, we present a comprehensive AC proteome and identified 113 putative AC proteins. The methods and protein inventories provide a framework for studying the biogenesis and modification of LROs and the mechanisms by which they participate in regulating metal ion metabolism.

USP25-PKM2-glycolysis axis contributes to ischemia reperfusion-induced acute kidney injury by promoting M1-like macrophage polarization and proinflammatory response.

M1-like macrophages have been reported to play critical roles in acute kidney injury (AKI). Here, we elucidated the role of ubiquitin-specific protease 25 (USP25) in M1-like macrophages polarization and AKI. High USP25 expression was correlated with a decline in renal function in patients with acute kidney tubular injury and in mice with AKI. In contrast, USP25 knockout reduced M1-like macrophage infiltration, suppressed M1-like polarization, and improved AKI in mice, indicating that USP25 was necessary for M1-like polarization and proinflammatory response. Immunoprecipitation assay and liquid chromatography-tandem mass spectrometry showed that the M2 isoform of pyruvate kinase, muscle (PKM2) was a target substrate of USP25. Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated the USP25 regulated aerobic glycolysis and lactate production during M1-like polarization via PKM2. Further analysis showed that the USP25-PKM2-aerobic glycolysis axis positively regulated M1-like polarization and exacerbated AKI in mice, providing potential therapeutic targets for AKI treatment.

Overexpression of the sulfate transporter-encoding SULTR2 increases chromium accumulation in Chlamydomonas reinhardtii.

Hexavalent chromium [Cr(Ⅵ)] is a highly toxic contaminant in aquatic systems, and microalgae represent promising bioremediators of metal-containing wastewater. However, the metal-binding capacity of algal cells is limited. Therefore, we improved the cellular Cr(Ⅵ) biosorption capacity of Chlamydomonas reinhardtii by overexpressing the sulfate transporter gene SULTR2. SULTR2 was predominantly located in the cytoplasm of the cell, and few proteins mobilized to the cell membrane as a Cr transporter under Cr stress conditions. Intracellular Cr accumulation was almost doubled in SULTR2-overexpressing transgenic strains after exposure to 30 µM K2 Cr2 O7 for 4 d. Alginate-based immobilization increased the rate of Cr removal from 43.81% to 88.15% for SULTR2-overexpressing transgenic strains after exposure to 10 µM K2 Cr2 O7 for 6 d. The immobilized cells also displayed a significant increase in nutrient removal efficiency compared to that of free-swimming cells. Therefore, SULTR2 overexpression in algae has a great potential for the bioremediation of Cr(Ⅵ)-containing wastewater.

Design and Experimental Study of a Large Beam Waist Streak Tube in an ICF Experiment.

In order to realize in situ multi-frame framing, this paper designed and developed a large-waist framing converter tube. The size ratio between the waist and the object was about 1.16:1. The subsequent test results showed that the static spatial resolution of the tube could reach 10 lp/mm (@ 72.5%) under the premise of this adjustment, and the transverse magnification could reach 2.9. Once the MCP (Micro Channel Plate) traveling wave gating unit is equipped at the output end, it is expected to promote the further development of in situ multi-frame framing technology.

[Preparation of two tanshinone â ¡_A-astragaloside â £ co-loaded nano-delivery systems and in vitro antitumor activity comparison].

This study screened excellent carriers for co-loading tanshinone Ⅱ_A(TSA) and astragaloside Ⅳ(As) to construct antitumor nano-drug delivery systems for TSA and As. TSA-As microemulsions(TSA-As-MEs) were prepared by water titration. TSA-As metal-organic framework(MOF) nano-delivery system was prepared by loading TSA and As in MOF by the hydrothermal method. Dynamic light scattering(DLS), transmission electron microscopy(TEM), and scanning electron microscopy(SEM) were used to characterize the physicochemical properties of the two preparations. Drug loading was determined by HPLC and the effects of the two preparations on the proliferation of vascular endothelial cells, T lymphocytes, and hepatocellular carcinoma cells were detected by the CCK-8 method. The results showed that the particle size, Zeta potential, and drug loading of TSA-As-MEs were(47.69±0.71) nm,(-14.70±0.49) mV, and(0.22±0.01)%, while those of TSA-As-MOF were(258.3±25.2) nm,(-42.30 ± 1.27) mV, and 15.35%±0.01%. TSA-As-MOF was superior to TSA-As-MEs in drug loading, which could inhibit the proliferation of bEnd.3 cells at a lower concentration and improve the proliferation ability of CTLL-2 cells significantly. Therefore, MOF was preferred as an excellent carrier for TSA and As co-loading.

Association between NAD+ levels and anaemia among women in community-based study.

Nicotinamide adenine dinucleotide (NAD+ ) level is the protective factor of cardiovascular diseases (CVDs). In addition, anaemia is a risk factor of adverse cardiovascular outcomes in women. However, there are limited data about the association between NAD+ and anaemia. The aim of this study was to evaluate association of NAD+ with anaemia among women. A total of 727 females from Jidong community were included in the current analysis. NAD+ levels were tested by the cycling assay and HPLC assay using whole blood samples. Anaemia was determined by haemoglobin (Hb) concentration, and the subtypes of anaemia were further defined according to mean corpuscular volume (MCV) in blood. Multivariable logistic analysis was used to analyse the association between NAD+ levels and anaemia or its subtypes. The mean age of recruited subjects was 42.7 years. The proportion of anaemia by NAD+ levels quartiles were 19.7% (35/178), 4.8% (9/189), 3.4% (6/178) and 2.7% (5/182). Haematological parameters including haemoglobin (Hb), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC) and red blood count (RBC) increased over NAD+ quartiles. Red cell volume distribution width (RDW) decreased over NAD+ quartiles. Compared with the lowest quartile of NAD+ levels (<27.6µM), the adjusted odds ratios with 95% confidence intervals of the top quartile were 0.15 (0.06-0.41) for anaemia, 0.05 (0.01-0.36) for microcytic anaemia and 0.37 (0.10-1.36) for normocytic anaemia respectively. Higher NAD+ levels were significantly associated with lower prevalence of anaemia among women, especially microcytic anaemia and normocytic anaemia. Haematological parameters might serve as a predictor of the blood NAD+ levels.

Effects of Serine/Arginine Enriched Protein BmUP on the Development of Male Silkworm Reproductive Organs.

Serine/arginine-rich proteins are a class of highly conserved splicing factor proteins involved in constitutive and alternative splicing. We screened a low molecular weight serine/arginine rich protein from silkworms and named it BmUP. Temporal and spatial expression analysis indicated that the BmUP gene was specifically expressed in the silkworm testis, and the highest expression occurred in the pre-pupa stage from the fifth instar to the moth stages. Here, we generated BmUP knockout individuals with the CRISPR/Cas9 system. Both the internal and external genitalia of knockout individuals were abnormal in knockout compared with wild-type male silkworms. In transgenic silkworms overexpressing BmUP, male silkworms showed a phenotype similar to that of the knockout individuals, whereas female individuals showed no significant differences from the wild type. In addition, by conducting promoter analysis, we identified Bmachi, a transcription factor that regulates the BmUP gene. Gel migration experiments revealed that BmAchi specifically binds the BmUP promoter. Quantitative real-time PCR showed that an increase in Bmachi expression up-regulated the expression of BmUP. In contrast, when the expression of Bmachi decreased, the expression of BmUP also downregulated in the experimental group compared with the control group. These results provide new insights for studying the effects of serine/arginine-rich proteins on the development of silkworm genitals.

Post-vaccination serologic testing of infants born to hepatitis B surface antigen-positive mothers is more cost-effective in Zhejiang Province, China: A Markov chain analysis.

To evaluate the cost-effectiveness of the post-vaccination serologic testing (PVST) plus active-passive immunoprophylaxis of infants born to hepatitis B surface antigen (HBsAg)-positive mothers, a Markov model was constructed by R 4.0.1 to compare the current strategy (three-dose HepB plus HBIG) and the PVST strategy (post-vaccination serologic testing plus the current strategy) for infants of HBsAg-positive mothers. Costs and utility scores were assessed from a field survey. Other model inputs were extracted from published literature and unpublished data from the Zhejiang provincial center for disease control and prevention (Zhejiang CDC). We calculated the incremental cost-effectiveness ratio (ICER) as the main result within 1-year cycle length with a 81 horizon among 50,000 infants and performed one-way sensitivity analysis and probabilistic sensitivity analysis to explore the reliability of outcome. The ICER was -4130.18 yuan/quality-adjusted life year (QALY) for the PVST strategy compared with the current strategy from the societal perspective. It was estimated that the PVST strategy would save about 3,809,546 yuan and prevent loss of 922.37 QALYs within 81 cycles among 50,000 infants. ICER was most sensitive to the discount rate, and the cost-effectiveness acceptability curves showed that the PVST strategy reached a probability of being 100% cost-effective below willing to pay (107,624 yuan). In conclusion, the PVST strategy had increased the utility and reduced cost among infants born to HBsAg-positive mothers. The PVST strategy is a more cost-effective choice for infants born to HBsAg-positive mothers than the current strategy, and further promotion of the PVST project is recommended.

Machine learning of plasma metabolome identifies biomarker panels for metabolic syndrome: findings from the China Suboptimal Health Cohort.

BACKGROUND: Metabolic syndrome (MetS) has been proposed as a clinically identifiable high-risk state for the prediction and prevention of cardiovascular diseases and type 2 diabetes mellitus. As a promising "omics" technology, metabolomics provides an innovative strategy to gain a deeper understanding of the pathophysiology of MetS. The study aimed to systematically investigate the metabolic alterations in MetS and identify biomarker panels for the identification of MetS using machine learning methods. METHODS: Nuclear magnetic resonance-based untargeted metabolomics analysis was performed on 1011 plasma samples (205 MetS patients and 806 healthy controls). Univariate and multivariate analyses were applied to identify metabolic biomarkers for MetS. Metabolic pathway enrichment analysis was performed to reveal the disturbed metabolic pathways related to MetS. Four machine learning algorithms, including support vector machine (SVM), random forest (RF), k-nearest neighbor (KNN), and logistic regression were used to build diagnostic models for MetS. RESULTS: Thirteen significantly differential metabolites were identified and pathway enrichment revealed that arginine, proline, and glutathione metabolism are disturbed metabolic pathways related to MetS. The protein-metabolite-disease interaction network identified 38 proteins and 23 diseases are associated with 10 MetS-related metabolites. The areas under the receiver operating characteristic curve of the SVM, RF, KNN, and logistic regression models based on metabolic biomarkers were 0.887, 0.993, 0.914, and 0.755, respectively. CONCLUSIONS: The plasma metabolome provides a promising resource of biomarkers for the predictive diagnosis and targeted prevention of MetS. Alterations in amino acid metabolism play significant roles in the pathophysiology of MetS. The biomarker panels and metabolic pathways could be used as preventive targets in dealing with cardiometabolic diseases related to MetS.

Polyubiquitylation of α-tubulin at K304 is required for flagellar disassembly in Chlamydomonas.

Cilia/flagella are structurally conserved and dynamic organelles; their assembly and disassembly are coordinated with the cell cycle and cell differentiation. Several post-translational modifications, including acetylation, methylation, phosphorylation and ubiquitylation, participate in ciliary disassembly. However, the detailed mechanism and the role of ubiquitylation in ciliary disassembly are unclear. This study identified 20 proteins that were ubiquitylated in shortening flagella of Chlamydomonas α-Tubulin was the most abundant ubiquitylated protein and it was labeled with K63 polyubiquitin chains primarily at K304. Expression of an α-tubulin mutant (K304R), which could not be ubiquitylated, decreased the rate of flagellar disassembly and resulted in an enrichment of the mutant form in the axoneme, suggesting that ubiquitylation of α-tubulin is required for the normal kinetics of axonemal disassembly. Immunoprecipitation and glutathione-S-transferase pulldown assays demonstrated that the retrograde intraflagellar transport (IFT) protein, IFT139, interacted with a variety of ubiquitylated proteins, including α-tubulin, suggesting that IFT-A was responsible for transporting ubiquitylated proteins out of the flagella. Our data suggest an important role for ubiquitylation and retrograde IFT in ciliary disassembly.This article has an associated First Person interview with the first author of the paper.

Influencing factors and necessity of post-vaccination serologic testing follow-up for HBsAg-positive mothers and their infants: A 5-year prospective study in Zhejiang Province, China (2016-2020).

In 2016, China officially proposed for the first time that infants born to HBsAg-positive mothers should be tested for HBsAg and anti-HBs 1-2 months after their third dose of HepB, also known as the post-vaccination serological testing programme. This study aimed to systematically evaluate the implementation and influencing factors of PVST to further reduce HBV infection risk in infants and improve the protective effect of HepB to the greatest extent. A prospective observational study was conducted to investigate the interruption of MTCT of hepatitis B. Univariate and multivariate analyses were applied to explore factors associated with the PVST follow-up rate among HBsAg-positive mothers and their infants. Additionally, bivariate analysis was performed on HBsAg and anti-HBs results in infants born to HBsAg-positive mothers. Here, the participation rate of PVST was 67.08% among 2120 pairs of positive mothers and infants. The HBsAg-positive rate among participants was 0.77%, whereas the anti-HBs positive rate was 96.84%, and both negative rates were 2.39%. Among infants with double negative results (34), only 15 completed three doses of HepB and PVST again, and 14 (93.33%) of which the antibody test results turned positive. Older mothers with high educational levels who reside in the local area were the most likely to PVST follow-up. The PVST programme is necessary to evaluate the HepB response status of newborns after vaccination. Moreover, revaccination for susceptible infants can effectively improve the MTCT-blocking rate of hepatitis B. Therefore, the scope of PVST projects in Zhejiang and China should be expanded.

Myopic Retinal Changes Screening: Comparison of Sensitivity and Specificity among 15 Combinations of Ultrawide Field Scanning Laser Ophthalmoscopy Images.

AIM: The objective of the study was to investigate the evaluation indices (diagnostic test accuracy and agreement) of 15 combinations of ultrawide field scanning laser ophthalmoscopy (UWF SLO) images in myopic retinal changes (MRC) screening to determine the combination of imaging that yields the highest evaluation indices in screening MRC. METHODS: This is a retrospective study of UWF SLO images obtained from myopes and were analyzed by 2 retinal specialists independently. Five field UWF SLO images that included the posterior (B), superior (S), inferior (I), nasal (N), and temporal (T) regions were obtained for analysis and its results used as a reference standard. The evaluation indices of different combinations comprising 1 to 4 fields of the retina were compared to determine the abilities of each combination screens for MRC. RESULTS: UWF SLO images obtained from 823 myopic patients (1,646 eyes) were included for the study. Sensitivities ranged from 50.0 to 98.9% (95% confidence interval (CI), 43.8-99.7%); the combinations of B + S + I (97.3%; 95% CI, 94.4-98.8%), B + T + S + I (98.5%; 95% CI, 95.9-99.5%), and B + S + N + I (98.9%; 95% CI, 96.4-99.7%) ranked highest. Furthermore, the combinations of B + S + I, B + T + S + I, and B + S + N + I also revealed the highest accuracy (97.7%; 95% CI, 95.1-100.0, 98.6; 95% CI, 96.7-100.0, 98.8; 95% CI, 96.9-100.0%) and agreement (kappa = 0.968, 0.980, and 0.980). For the various combinations, specificities were all higher than 99.5% (95% CI, 99.3-100.0%). CONCLUSIONS: In our study, screening combinations of B + S + I, B + T + S + I, and B + S + N + I stand out with high-performing optimal evaluation indices. However, when time is limited, B + S + I may be more applicable in primary screening of MRC.

[Preparation of hydrophilic Indigo Naturalis based on polyethylene glycol( PEG) surface film-forming method and evaluation].

Polyethylene glycol (PEG) surface film-forming method was used to prepare hydrophilic Indigo Naturalis decoction pieces with stable effect.The preparation process of modified Indigo Naturalis was optimized and its microscopic properties,hydrophilicity,antipyretic efficacy,and safety were systematically evaluated.With equilibrium contact angle as assessment index,the influence of modifier type,modifier dosage,dispersant dosage,and co-grinding time on water solubility of Indigo Naturalis was investigated by single factor test.The results showed that the optimal preparation process was as follows.The 6%PEG6000 is dissolved in 10%anhydrous ethanol solution by sonification and then the mixture is ground with Indigo Naturalis for 2 min.The resultant product is dried on a square tray in an oven at 60℃to remove ethanol and thereby the PEG-modified hydrophilic Indigo Naturalis decoction pieces are yielded.The morphological observation under scanning electron microscope (SEM) indicated that the modified Indigo Naturalis had smoother surface than Indigo Naturalis,and energy spectrometer measurement showed that the nitrogen (N),calcium(Ca),oxygen (O),and silicon (Si) on the surface of modified Indigo Naturalis powder were less than those of Indigo Naturalis powder.Modified Indigo Naturalis had the equilibrium contact angle 18.96°smaller,polar component 22.222 m J·m~(-2)more,and nonpolar component 7.277 m J·m~(-2)smaller than the Indigo Naturalis powder.Multiple light scattering technique was employed to evaluate the dispersion in water and the result demonstrated that the transmittance of Indigo Naturalis and modified Indigo Naturalis was about85%and 75%,respectively,suggesting the higher dispersity of modified Indigo Naturalis.The suspension rate of modified Indigo Naturalis in water was determined by reflux treatment.The result showed that 57%of Indigo Naturalis was not wetted after refluxing for1 h,while the modified Indigo Naturalis was all wetted and dispersed into water.The dissolution of indigo and indirubin of modified Indigo Naturalis increased and the process was more stable.Then,rats were randomized into the blank group,model group,acetaminophen group,Indigo Naturalis group,and hydrophilic Indigo Naturalis group.The temperature changes of rats were observed after administration and the concentration of IL-1ßand TNF-αin serum and IL-1ßand PGE_2in hypothalamus was measured.The results indicated that the temperature of Indigo Naturalis group and hydrophilic Indigo Naturalis group dropped and the IL-1ßlevel of the hydrophilic Indigo Naturalis group decreased (P<0.05) as compared with those in the model group.Thus,both Indigo Naturalis and hydrophilic Indigo Naturalis had antipyretic effect,particularly the hydrophilic Indigo Naturalis.The acute toxicity test of hydrophilic Indigo Naturalis verified that it had no toxicity to rats.In this study,the hydrophilic Indigo Naturalis decoction pieces were prepared with the PEG surface film-forming method,and the antipyretic efficacy and safety were evaluated,which expanded the technological means of powder modification for Chinese medicine and provided a method for clinical use of Chinese medicine.

Fis Contributes to Resistance of Pseudomonas aeruginosa to Ciprofloxacin by Regulating Pyocin Synthesis.

Factor for inversion stimulation (Fis) is a versatile DNA binding protein that plays an important role in coordinating bacterial global gene expression in response to growth phases and environmental stresses. Previously, we demonstrated that Fis regulates the type III secretion system (T3SS) in Pseudomonas aeruginosa In this study, we explored the role of Fis in the antibiotic resistance of P. aeruginosa and found that mutation of the fis gene increases the bacterial susceptibility to ciprofloxacin. We further demonstrated that genes related to pyocin biosynthesis are upregulated in the fis mutant. The pyocins are produced in response to genotoxic agents, including ciprofloxacin, and the release of pyocins results in lysis of the producer cell. Thus, pyocin biosynthesis genes sensitize P. aeruginosa to ciprofloxacin. We found that PrtN, the positive regulator of the pyocin biosynthesis genes, is upregulated in the fis mutant. Genetic experiments and electrophoretic mobility shift assays revealed that Fis directly binds to the promoter region of prtN and represses its expression. Therefore, our results revealed novel Fis-mediated regulation on pyocin production and bacterial resistance to ciprofloxacin in P. aeruginosaIMPORTANCEPseudomonas aeruginosa is an important opportunistic pathogenic bacterium that causes various acute and chronic infections in human, especially in patients with compromised immunity, cystic fibrosis (CF), and/or severe burn wounds. About 60% of cystic fibrosis patients have a chronic respiratory infection caused by P. aeruginosa The bacterium is intrinsically highly resistant to antibiotics, which greatly increases difficulties in clinical treatment. Therefore, it is critical to understand the mechanisms and the regulatory pathways that are involved in antibiotic resistance. In this study, we elucidated a novel regulatory pathway that controls the bacterial resistance to fluoroquinolone antibiotics, which enhances our understanding of how P. aeruginosa responds to ciprofloxacin.

Long noncoding RNA CCAL transferred from fibroblasts by exosomes promotes chemoresistance of colorectal cancer cells.

Long noncoding RNAs (lncRNAs) are involved in the pathology of colorectal cancer (CRC). Current efforts to eradicate CRC predominantly focused on targeting the proliferation of rapidly growing cancer epithelial cells. This is largely ineffective with resistance arising in most tumors after exposure to chemotherapy. Despite the long-standing recognition of the crosstalk between carcinoma-associated fibroblasts (CAFs) and cancer cells in the tumor microenvironment, how CAFs may contribute to drug resistance in neighboring cancer cells is not well characterized. Here, we show that lncRNA CCAL (colorectal cancer-associated lncRNA) promotes oxaliplatin (Oxa) resistance of CRC cells. RNA-ISH shows higher CCAL expressed in the tumor stroma compared to cancer nests of CRC tissues. Functional studies reveal that CCAL is transferred from CAFs to the cancer cells via exosomes, where it suppresses CRC cell apoptosis, confers chemoresistance and activates ß-catenin pathway in vitro and in vivo. Mechanistically, CCAL interacts directly with mRNA stabilizing protein HuR (human antigen R) to increase ß-catenin mRNA and protein levels. Our findings indicate that CCAL expressed by CAFs of the colorectal tumor stroma contributes to tumor chemoresistance and CCAL may serve as a potential therapeutic target for Oxa resistance.

Target-Induced Core-Satellite Nanostructure Assembly Strategy for Dual-Signal-On Fluorescence Imaging and Raman Quantification of Intracellular MicroRNA Guided Photothermal Therapy.

Integrating biological detection and treatment into one system is a smart therapeutic maneuver for efficient cancer treatment. Herein, a target-activated core-satellite nanostructure (CS nanostructure) assembly built on gold nanobipyramids motor (AuNBPs motor)/gold nanoparticle probe (AuNP probe) exhibiting simultaneous dual signal-on imaging, quantification of intracellular microRNA-21 (miR-21), and photothermal therapy (PTT) for cancer is designed. Of note, when the AuNBPs motor/AuNP probe enters into cells, miR-21 triggers the reaction between AuNBPs motor and AuNP probe, resulting in the formation of CS nanostructure assembly. The process of assembling the CS nanostructure is accompanied with strong fluorescent signals from TAMRA and surface-enhanced Raman scattering (SERS) signals from adenine. The fluorescent signal is leveraged to image the intracellular miR-21 level, whereas the SERS signal is utilized for absolute quantification of intracellular miR-21, and the CS nanostructure acts as the photosensitizer for PTT. This strategy can successfully image and quantify miR-21 in a single cell, and also distinguish normal cells from tumor cells. Moreover, under the guidance of fluorescence signal, the assembly kills tumor cells and inhibits tumor growth via PTT. In vitro and in vivo results prove that the proposed strategy possesses enormous potential for application in the diagnosis and treatment of cancer.

Stepwise progressive parametric multiple testing procedure with correlated normal test statistics.

Multiple testing problems are often seen in clinical trials. An appropriate testing procedure should be specified to deal with the potential inflation of type I error rate due to multiplicity. In this article, we propose a stepwise progressive parametric multiple (SPPM) testing procedure, which constructs the testing using the products of all the combinations of local [Formula: see text]-values and the critical values are determined by numerical integrations progressively using the closure principle. We have compared the performance of SPPM to several other procedures, and demonstrate the advantage of SPPM procedure, in terms of power, for the certain situations of multiple testing.

MiR-1246 promotes anti-apoptotic effect of mini-αA in oxidative stress-induced apoptosis in retinal pigment epithelial cells.

BACKGROUND: Geographic atrophy (GA) is a late-stage symptom of an age-related macular degeneration (AMD), characterized by the loss of retinal pigment epithelial (RPE) cells and photoreceptor functions. Despite being a major cause of blindness in individuals of 65 years of age and older, some forms of AMD, including GA, still lack targeted treatment. Our previous study demonstrated that mini-αA peptide, which contains the functional site of αA-crystallin, protected RPE cells from NaIO3 -induced apoptosis. METHODS: To further investigate the underlying mechanism, we applied next-generation sequencing analysis to identify miR-1246 as a putative mediator of mini-αA protective function. To investigate the role of miR-1246 in RPE cell apoptosis, a stable miR-1246-low-expression cell line was established by using miR-1246 inhibitor. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to investigate the proliferation of RPE cells, mRNA and miR-1246 expression were detected by the quantitative reverse transcriptase-polymerase chain reaction. RESULTS: We have further identified caspase-3 and caspase-14 as molecular targets of miR-1246 involved in regulation of apoptosis in NaIO3 -incubated cells. Interestingly, disruption of miR-1246 expression enhanced anti-apoptotic effect of mini-αA on RPE cells during oxidative stress. CONCLUSIONS: Our results provide a mechanistic basis for evaluation of miR-1246 as a new candidate target for the clinical treatment of AMD.