The repertoire of mutational signatures in human cancer.

Somatic mutations in cancer genomes are caused by multiple mutational processes, each of which generates a characteristic mutational signature1. Here, as part of the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium2 of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA), we characterized mutational signatures using 84,729,690 somatic mutations from 4,645 whole-genome and 19,184 exome sequences that encompass most types of cancer. We identified 49 single-base-substitution, 11 doublet-base-substitution, 4 clustered-base-substitution and 17 small insertion-and-deletion signatures. The substantial size of our dataset, compared with previous analyses3-15, enabled the discovery of new signatures, the separation of overlapping signatures and the decomposition of signatures into components that may represent associated-but distinct-DNA damage, repair and/or replication mechanisms. By estimating the contribution of each signature to the mutational catalogues of individual cancer genomes, we revealed associations of signatures to exogenous or endogenous exposures, as well as to defective DNA-maintenance processes. However, many signatures are of unknown cause. This analysis provides a systematic perspective on the repertoire of mutational processes that contribute to the development of human cancer.

Genomic editing of metformin efficacy-associated genetic variants in SLC47A1 does not alter SLC47A1 expression.

Several pharmacogenetics studies have identified an association between a greater metformin-dependent reduction in HbA1c levels and the minor A allele at rs2289669 in intron 10 of SLC47A1, encoding multidrug and toxin extrusion 1 (MATE1), a presumed metformin transporter. It is currently unknown if the rs2289669 locus is a cis-eQTL, which would validate its role as predictor of metformin efficacy. We looked at association between common genetic variants in the SLC47A1 gene region and HbA1c reduction after metformin treatment using locus-wise meta-analysis from the MetGen consortium. CRISPR-Cas9 was applied to perform allele editing of, or genomic deletion around, rs2289669 and of the closely linked rs8065082 in HepG2 cells. The genome-edited cells were evaluated for SLC47A1 expression and splicing. None of the common variants including rs2289669 showed significant association with metformin response. Genomic editing of either rs2289669 or rs8065082 did not alter SLC47A1 expression or splicing. Experimental and in silico analyses show that the rs2289669-containing haploblock does not appear to carry genetic variants that could explain its previously reported association with metformin efficacy.

Iron efflux by IetA enhances ß-lactam aztreonam resistance and is linked to oxidative stress through cellular respiration in Riemerella anatipestifer.

BACKGROUND: Riemerella anatipestifer encodes an iron acquisition system, but whether it encodes the iron efflux pump and its role in antibiotic resistance are largely unknown. OBJECTIVES: To screen and identify an iron efflux gene in R. anatipestifer and determine whether and how the iron efflux gene is involved in antibiotic resistance. METHODS: In this study, gene knockout, streptonigrin susceptibility assay and inductively coupled plasma mass spectrometry were used to screen for the iron efflux gene ietA. The MIC measurements, scanning electron microscopy and reactive oxygen species (ROS) detection were used to verify the role of IetA in aztreonam resistance and its mechanism. Mortality and colonization assay were used to investigate the role of IetA in virulence. RESULTS: The deletion mutant ΔietA showed heightened susceptibility to streptonigrin, and prominent intracellular iron accumulation was observed in ΔfurΔietA under excess iron conditions. Additionally, ΔietA exhibited increased sensitivity to H2O2-produced oxidative stress. Under aerobic conditions with abundant iron, ΔietA displayed increased susceptibility to the ß-lactam antibiotic aztreonam due to heightened ROS production. However, the killing efficacy of aztreonam was diminished in both WT and ΔietA under anaerobic or iron restriction conditions. Further experiments demonstrated that the efficiency of aztreonam against ΔietA was dependent on respiratory complexes Ⅰ and Ⅱ. Finally, in a duckling model, ΔietA had reduced virulence compared with the WT. CONCLUSION: Iron efflux is critical to alleviate oxidative stress damage and ß-lactam aztreonam killing in R. anatipestifer, which is linked by cellular respiration.

AZD7762 induces CRBN dependent BAG3 degradation through ubiquitin-proteasome pathway.

Protein degraders are currently under rapid development as a promising modality for drug discovery. They are compounds that orchestrate interactions between a target protein and an E3 ubiquitin ligase, prompting intracellular protein degradation through proteasomal pathway. More protein degraders identification will greatly promote the development of this field. BAG3 is widely recognized as an excellent therapeutic target in cancer treatments. Exploring protein degraders that target BAG3 degradation has profound implications. Herein, molecular docking was applied to assess binding energy between 81 clinical phase I kinase inhibitors and BAG3. BAG3 protein and mRNA level were detected by western blot and quantitative real-time PCR. CCK8 assay and colony formation assay were applied to detect the cell viability and proliferation rate. Cell death was accessed using flow cytometry combined with PI and Annexin V double staining. AZD7762, a Chk1 kinase inhibitor, was identified to induce BAG3 degradation in a ubiquitin-proteasome pathway. AZD7762-induced BAG3 degradation was not dependent on Chk1 expression or activity. CRBN, an E3 ligase, was identified to bind to BAG3 and mediated BAG3 ubiquitination in the presence of AZD7762. By targeting Chk1 and BAG3, two ideal therapeutic targets in cancer treatment, AZD7762 would be a powerful chemotherapy agent in the future.

Engineered allele substitution at PPARGC1A rs8192678 alters human white adipocyte differentiation, lipogenesis, and PGC-1α content and turnover.

AIMS/HYPOTHESIS: PPARGC1A encodes peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α), a central regulator of energy metabolism and mitochondrial function. A common polymorphism in PPARGC1A (rs8192678, C/T, Gly482Ser) has been associated with obesity and related metabolic disorders, but no published functional studies have investigated direct allele-specific effects in adipocyte biology. We examined whether rs8192678 is a causal variant and reveal its biological function in human white adipose cells. METHODS: We used CRISPR-Cas9 genome editing to perform an allelic switch (C-to-T or T-to-C) at rs8192678 in an isogenic human pre-adipocyte white adipose tissue (hWAs) cell line. Allele-edited single-cell clones were expanded and screened to obtain homozygous T/T (Ser482Ser), C/C (Gly482Gly) and heterozygous C/T (Gly482Ser) isogenic cell populations, followed by functional studies of the allele-dependent effects on white adipocyte differentiation and mitochondrial function. RESULTS: After differentiation, the C/C adipocytes were visibly less BODIPY-positive than T/T and C/T adipocytes, and had significantly lower triacylglycerol content. The C allele presented a dose-dependent lowering effect on lipogenesis, as well as lower expression of genes critical for adipogenesis, lipid catabolism, lipogenesis and lipolysis. Moreover, C/C adipocytes had decreased oxygen consumption rate (OCR) at basal and maximal respiration, and lower ATP-linked OCR. We determined that these effects were a consequence of a C-allele-driven dysregulation of PGC-1α protein content, turnover rate and transcriptional coactivator activity. CONCLUSIONS/INTERPRETATION: Our data show allele-specific causal effects of the rs8192678 variant on adipogenic differentiation. The C allele confers lower levels of PPARGC1A mRNA and PGC-1α protein, as well as disrupted dynamics of PGC-1α turnover and activity, with downstream effects on cellular differentiation and mitochondrial function. Our study provides the first experimentally deduced insights on the effects of rs8192678 on adipocyte phenotype.

Manganese Efflux Achieved by MetA and MetB Affects Oxidative Stress Resistance and Iron Homeostasis in Riemerella anatipestifer.

In bacteria, manganese homeostasis is controlled by import, regulation, and efflux. Here, we identified 2 Mn exporters, MetA and MetB (manganese efflux transporters A and B), in Riemerella anatipestifer CH-1, encoding a putative cation diffusion facilitator (CDF) protein and putative resistance-nodulation-division (RND) efflux pump, respectively. Compared with the wild type (WT), ΔmetA, ΔmetB, and ΔmetAΔmetB exhibited sensitivity to manganese, since they accumulated more intracellular Mn2+ than the WT under excess manganese conditions, while the amount of iron in the mutants was decreased. Moreover, ΔmetA, ΔmetB, and ΔmetAΔmetB were more sensitive to the oxidant NaOCl than the WT. Further study showed that supplementation with iron sources could alleviate manganese toxicity and that excess manganese inhibited bacterial cell division. RNA-Seq showed that manganese stress resulted in the perturbation of iron metabolism genes, further demonstrating that manganese efflux is critical for iron homeostasis. metA transcription was upregulated under excess manganese but was not activated by MetR, a DtxR family protein, although MetR was also involved in manganese detoxification, while metB transcription was downregulated under iron depletion conditions and in fur mutants. Finally, homologues of MetA and MetB were found to be mainly distributed in members of Flavobacteriaceae. Specifically, MetB represents a novel manganese exporter in Gram-negative bacteria. IMPORTANCE Manganese is required for the function of many proteins in bacteria, but in excess, manganese can mediate toxicity. Therefore, the intracellular levels of manganese must be tightly controlled. Manganese efflux transporters have been characterized in some other bacteria; however, their homologues could not be found in the genome of Riemerella anatipestifer through sequence comparison. This indicated that other types of manganese efflux transporters likely exist. In this study, we characterized 2 transporters, MetA and MetB, that mediate manganese efflux in R. anatipestifer in response to manganese overload. MetA encodes a putative cation diffusion facilitator (CDF) protein, which has been characterized as a manganese transporter in other bacteria, while this is the first observation of a putative resistance-nodulation-division (RND) transporter contributing to manganese export in Gram-negative bacteria. In addition, the mechanism of manganese toxicity was studied by observing morphological changes and by transcriptome sequencing. Taken together, these results are important for expanding our understanding of manganese transporters and revealing the mechanism of manganese toxicity.

In-depth characterization of the cisplatin mutational signature in human cell lines and in esophageal and liver tumors.

Cisplatin reacts with DNA and thereby likely generates a characteristic pattern of somatic mutations, called a mutational signature. Despite widespread use of cisplatin in cancer treatment and its role in contributing to secondary malignancies, its mutational signature has not been delineated. We hypothesize that cisplatin's mutational signature can serve as a biomarker to identify cisplatin mutagenesis in suspected secondary malignancies. Knowledge of which tissues are at risk of developing cisplatin-induced secondary malignancies could lead to guidelines for noninvasive monitoring for secondary malignancies after cisplatin chemotherapy. We performed whole genome sequencing of 10 independent clones of cisplatin-exposed MCF-10A and HepG2 cells and delineated the patterns of single and dinucleotide mutations in terms of flanking sequence, transcription strand bias, and other characteristics. We used the mSigAct signature presence test and nonnegative matrix factorization to search for cisplatin mutagenesis in hepatocellular carcinomas and esophageal adenocarcinomas. All clones showed highly consistent patterns of single and dinucleotide substitutions. The proportion of dinucleotide substitutions was high: 8.1% of single nucleotide substitutions were part of dinucleotide substitutions, presumably due to cisplatin's propensity to form intra- and interstrand crosslinks between purine bases in DNA. We identified likely cisplatin exposure in nine hepatocellular carcinomas and three esophageal adenocarcinomas. All hepatocellular carcinomas for which clinical data were available and all esophageal cancers indeed had histories of cisplatin treatment. We experimentally delineated the single and dinucleotide mutational signature of cisplatin. This signature enabled us to detect previous cisplatin exposure in human hepatocellular carcinomas and esophageal adenocarcinomas with high confidence.

An Exposed Outer Membrane Hemin-Binding Protein Facilitates Hemin Transport by a TonB-Dependent Receptor in Riemerella anatipestifer.

Iron is an essential element for the replication of most bacteria, including Riemerella anatipestifer, a Gram-negative bacterial pathogen of ducks and other birds. R. anatipestifer utilizes hemoglobin-derived hemin as an iron source; however, the mechanism by which this bacterium acquires hemin from hemoglobin is largely unknown. Here, rhuA disruption was shown to impair iron utilization from duck hemoglobin in R. anatipestifer CH-1. Moreover, the putative lipoprotein RhuA was identified as a surface-exposed, outer membrane hemin-binding protein, but it could not extract hemin from duck hemoglobin. Mutagenesis studies showed that recombinant RhuAY144A, RhuAY177A, and RhuAH149A lost hemin-binding ability, suggesting that amino acid sites at tyrosine 144 (Y144), Y177, and histidine 149 (H149) are crucial for hemin binding. Furthermore, rhuR, the gene adjacent to rhuA, encodes a TonB2-dependent hemin transporter. The function of rhuA in duck hemoglobin utilization was abolished in the rhuR mutant strain, and recombinant RhuA was able to bind the cell surface of R. anatipestifer CH-1 ΔrhuA rather than R. anatipestifer CH-1 ΔrhuR ΔrhuA, indicating that RhuA associates with RhuR to function. The sequence of the RhuR-RhuA hemin utilization locus exhibits no similarity to those of characterized hemin transport systems. Thus, this locus is a novel hemin uptake locus with homologues distributed mainly in the Bacteroidetes phylum. IMPORTANCE In vertebrates, hemin from hemoglobin is an important iron source for infectious bacteria. Many bacteria can obtain hemin from hemoglobin, but the mechanisms of hemin acquisition from hemoglobin differ among bacteria. Moreover, most studies have focused on the mechanism of hemin acquisition from mammalian hemoglobin. In this study, we found that the RhuR-RhuA locus of R. anatipestifer CH-1, a duck pathogen, is involved in hemin acquisition from duck hemoglobin via a unique pathway. RhuA was identified as an exposed outer membrane hemin-binding protein, and RhuR was identified as a TonB2-dependent hemin transporter. Moreover, the function of RhuA in hemoglobin utilization is RhuR dependent and not vice versa. The homologues of RhuR and RhuA are widely distributed in bacteria in marine environments, animals, and plants, representing a novel hemin transportation system of Gram-negative bacteria. This study not only was important for understanding hemin uptake in R. anatipestifer but also enriched the knowledge about the hemin transportation pathway in Gram-negative bacteria.

Functional characterization of Fur in iron metabolism, oxidative stress resistance and virulence of Riemerella anatipestifer.

Iron is essential for most bacteria to survive, but excessive iron leads to damage by the Fenton reaction. Therefore, the concentration of intracellular free iron must be strictly controlled in bacteria. Riemerella anatipestifer (R. anatipestifer), a Gram-negative bacterium, encodes the iron uptake system. However, the iron homeostasis mechanism remains largely unknown. In this study, it was shown that compared with the wild type R. anatipestifer CH-1, R. anatipestifer CH-1Δfur was more sensitive to streptonigrin, and this effect was alleviated when the bacteria were cultured in iron-depleted medium, suggesting that the fur mutant led to excess iron accumulation inside cells. Similarly, compared with R. anatipestifer CH-1∆recA, R. anatipestifer CH-1∆recAΔfur was more sensitive to H2O2-induced oxidative stress when the bacteria were grown in iron-rich medium rather than iron-depleted medium. Accordingly, it was shown that R. anatipestifer CH-1∆recAΔfur produced more intracellular ROS than R. anatipestifer CH-1∆recA in iron-rich medium. Electrophoretic mobility shift assays showed that R. anatipestifer CH-1 Fur suppressed the transcription of putative iron uptake genes through binding to their promoter regions. Finally, it was shown that compared with the wild type, R. anatipestifer CH-1Δfur was significantly attenuated in ducklings and that the colonization ability of R. anatipestifer CH-1Δfur in various tissues or organs was decreased. All these results suggested that Fur is important for iron homeostasis in R. anatipestifer and its pathogenic mechanism.

Genome-scale mutational signatures of aflatoxin in cells, mice, and human tumors.

Aflatoxin B1 (AFB1) is a mutagen and IARC (International Agency for Research on Cancer) Group 1 carcinogen that causes hepatocellular carcinoma (HCC). Here, we present the first whole-genome data on the mutational signatures of AFB1 exposure from a total of >40,000 mutations in four experimental systems: two different human cell lines, in liver tumors in wild-type mice, and in mice that carried a hepatitis B surface antigen transgene-this to model the multiplicative effects of aflatoxin exposure and hepatitis B in causing HCC. AFB1 mutational signatures from all four experimental systems were remarkably similar. We integrated the experimental mutational signatures with data from newly sequenced HCCs from Qidong County, China, a region of well-studied aflatoxin exposure. This indicated that COSMIC mutational signature 24, previously hypothesized to stem from aflatoxin exposure, indeed likely represents AFB1 exposure, possibly combined with other exposures. Among published somatic mutation data, we found evidence of AFB1 exposure in 0.7% of HCCs treated in North America, 1% of HCCs from Japan, but 16% of HCCs from Hong Kong. Thus, aflatoxin exposure apparently remains a substantial public health issue in some areas. This aspect of our study exemplifies the promise of future widespread resequencing of tumor genomes in providing new insights into the contribution of mutagenic exposures to cancer incidence.

Attosecond Time-Domain Measurement of Core-Level-Exciton Decay in Magnesium Oxide.

Excitation of ionic solids with extreme ultraviolet pulses creates localized core-level excitons, which in some cases couple strongly to the lattice. Here, core-level-exciton states of magnesium oxide are studied in the time domain at the Mg L_{2,3} edge with attosecond transient reflectivity spectroscopy. Attosecond pulses trigger the excitation of these short-lived quasiparticles, whose decay is perturbed by time-delayed near-infrared pulses. Combined with a few-state theoretical model, this reveals that the infrared pulse shifts the energy of bright (dipole-allowed) core-level-exciton states as well as induces features arising from dark core-level excitons. We report coherence lifetimes for the two lowest core-level excitons of 2.3±0.2 and 1.6±0.5 fs and show that these are primarily a consequence of strong exciton-phonon coupling, disclosing the drastic influence of structural effects in this ultrafast relaxation process.

New Perspectives on Galleria mellonella Larvae as a Host Model Using Riemerella anatipestifer as a Proof of Concept.

Galleria mellonella larvae have been used as a host model to study interactions between pathogens and hosts for several years. However, whether the model is useful to interrogate Riemerella anatipestifer infection biology remained unknown. This study aimed to exploit the potential of G. mellonella larvae and reveal their limitations as a host model for R. anatipestifer infection. G. mellonella larvae were shown to be effective for virulence evaluations of different R. anatipestifer strains. Furthermore, the virulent strain R. anatipestifer CH-1 had a stronger ability to proliferate than the attenuated strain R. anatipestifer ATCC 11845 in both G. mellonella larvae and ducklings. Unconventionally it was shown that G. mellonella larvae cannot be used to evaluate the efficacy of antimicrobials and their combinations. Additionally, it was shown that certain virulence factors, such as OmpA (B739_0861), B739_1208, B739_1343, and Wza (B739_1124), were specific only for ducklings, suggesting that G. mellonella larvae must be cautiously used to identify virulence factors of R. anatipestifer Evaluation of heme uptake-related virulence genes, such as tonB1 and tonB2, required preincubating the strains with hemoglobin before infection of G. mellonella larvae since R. anatipestifer cannot obtain a heme source from G. mellonella larvae. In conclusion, this study revealed the applicability and limitations of G. mellonella as a model with which to study the pathogen-host interaction, particularly in the context of R. anatipestifer infection.

SigProfilerMatrixGenerator: a tool for visualizing and exploring patterns of small mutational events.

BACKGROUND: Cancer genomes are peppered with somatic mutations imprinted by different mutational processes. The mutational pattern of a cancer genome can be used to identify and understand the etiology of the underlying mutational processes. A plethora of prior research has focused on examining mutational signatures and mutational patterns from single base substitutions and their immediate sequencing context. We recently demonstrated that further classification of small mutational events (including substitutions, insertions, deletions, and doublet substitutions) can be used to provide a deeper understanding of the mutational processes that have molded a cancer genome. However, there has been no standard tool that allows fast, accurate, and comprehensive classification for all types of small mutational events. RESULTS: Here, we present SigProfilerMatrixGenerator, a computational tool designed for optimized exploration and visualization of mutational patterns for all types of small mutational events. SigProfilerMatrixGenerator is written in Python with an R wrapper package provided for users that prefer working in an R environment. SigProfilerMatrixGenerator produces fourteen distinct matrices by considering transcriptional strand bias of individual events and by incorporating distinct classifications for single base substitutions, doublet base substitutions, and small insertions and deletions. While the tool provides a comprehensive classification of mutations, SigProfilerMatrixGenerator is also faster and more memory efficient than existing tools that generate only a single matrix. CONCLUSIONS: SigProfilerMatrixGenerator provides a standardized method for classifying small mutational events that is both efficient and scalable to large datasets. In addition to extending the classification of single base substitutions, the tool is the first to provide support for classifying doublet base substitutions and small insertions and deletions. SigProfilerMatrixGenerator is freely available at https://github.com/AlexandrovLab/SigProfilerMatrixGenerator with an extensive documentation at https://osf.io/s93d5/wiki/home/ .

Methyl 3,4-dihydroxybenzoate protects retina in a mouse model of acute ocular hypertension through multiple pathways.

Methyl 3,4 dihydroxybenzoate (MDHB) is a small molecule that shows neuroprotective effects in vitro and in a photoreceptor-degenerative mouse model. Here we investigated whether MDHB protects retina in a mouse model of acute ocular hypertension (AOH) and explores the underlying mechanisms. AOH was induced in mice by increasing intraocular pressure to approximately 90 mmHg for 60 min, then MDHB or vehicle was intraperitoneally injected daily up to 7 days. Immunostaining and multi-electrode array recordings were performed to examine the structure and function of retinas receiving the treatments. Western-blotting was applied to test the expression of several proteins related to oxidative stress and brain-derived neurotrophic factor (BDNF)-initiated signaling. Results showed that AOH injury reduced the number of Brn3a-stained retinal ganglion cells (RGCs) and ChAT-amacrine cells; thinned the inner retinal layers and induced apoptosis. Physiologically, AOH decreased the response of OFF and ON-OFF RGCs. All of these changes were reversed by MDHB-treatment. Mechanistically, MDHB appeared to work on three parallel pathways: (1) MDHB decreased the production of reactive oxygen species, the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and cytosol heme oxygenase 1 (HO-1); (2) It upregulated the expression of BDNF and its receptor tropomyosin-related kinase B (TrkB), and activated the downstream AKT pathways; (3) It inhibited reactive gliosis by reducing GFAP and Iba-1 expression. Thus our results suggest that MDHB protects retina against AOH injury by inhibiting oxidative stress, activating the BDNF/AKT signaling and inhibiting inflammatory pathways. Therefore, MDHB may serve as a promising candidate to treat retinal ischemia.

A study of positioning orientation effect on segmentation accuracy using convolutional neural networks for rectal cancer.

PURPOSE: Convolutional neural networks (CNN) have greatly improved medical image segmentation. A robust model requires training data can represent the entire dataset. One of the differing characteristics comes from variability in patient positioning (prone or supine) for radiotherapy. In this study, we investigated the effect of position orientation on segmentation using CNN. METHODS: Data of 100 patients (50 in supine and 50 in prone) with rectal cancer were collected for this study. We designed three sets of experiments for comparison: (a) segmentation using the model trained with data from the same orientation; (b) segmentation using the model trained with data from the opposite orientation; (c) segmentation using the model trained with data from both orientations. We performed fivefold cross-validation. The performance was evaluated on segmentation of the clinical target volume (CTV), bladder, and femurs with Dice similarity coefficient (DSC) and Hausdorff distance (HD). RESULTS: Compared with models trained on cases positioned in the same orientation, the models trained with cases positioned in the opposite orientation performed significantly worse (P < 0.05) on CTV and bladder segmentation, but had comparable accuracy for femurs (P > 0.05). The average DSC values were 0.74 vs 0.84, 0.85 vs 0.88, and 0.91 vs 0.91 for CTV, bladder, and femurs, respectively. The corresponding HD values (mm) were 16.6 vs 14.6, 8.4 vs 8.1, and 6.3 vs 6.3, respectively. The models trained with data from both orientations have comparable accuracy (P > 0.05), with average DSC of 0.84, 0.88, and 0.91 and HD of 14.4, 8.1, and 6.3, respectively. CONCLUSIONS: Orientation affects the accuracy for CTV and bladder, but has negligible effect on the femurs. The model trained from data combining both orientations performs as well as a model trained with data from the same orientation for all the organs. These observations can offer guidance on the choice of training data for accurate segmentation.

MicroRNA-132 upregulation promotes matrix degradation in intervertebral disc degeneration.

MicroRNAs (miRNAs) have been shown to be involved in the pathogenesis of intervertebral disc degeneration (IDD). This experiment was designed to study the expression and role of the miRNA, miR-132, in IDD. MiR-132 expression in human nucleus pulposus (NP) tissue was assessed by quantitative real-time PCR. The methylation status of the miR-132 was assessed with methylation-specific PCR and bisulfite sequencing PCR. The regulation of growth differentiation factor5 (GDF5) expression by miR-132 was evaluated by luciferase reporter assay. Moreover, we investigated the function of miR-132 on IDD in vivo using a classic needle-punctured rat tail model. These results showed that miR-132 expression was upregulated during IDD and this upregulation was associated with hypomethylation of its promoter. MiR-132 overexpression led to increased expression of ECM catabolic factors, including MMP13 and ADAMTS4, in NP cells while levels of anabolic proteins, such as type II collagen and aggrecan, were diminished. GDF5 was identified as a direct target of negative regulation by miR-132. MAPK/ERK signaling was also found to be associated with miR-132-induced ECM degradation. In addition, we showed that miR-132 inhibition effectively attenuated NP ECM degradation in IDD in vivo. Our findings demonstrated that miR-132 promotes ECM degradation by human NP cells by direct targeting of GDF5. Hence, miR-132 represents a potential therapeutic target in the treatment of IDD.

Knockout of RP2 decreases GRK1 and rod transducin subunits and leads to photoreceptor degeneration in zebrafish.

Retinitis pigmentosa (RP) affects about 1.8 million individuals worldwide. X-linked retinitis pigmentosa (XLRP) is one of the most severe forms of RP. Nearly 85% of XLRP cases are caused by mutations in the X-linked retinitis pigmentosa 2 (RP2) and RPGR. RP2 has been considered to be a GTPase activator protein for ARL3 and to play a role in the traffic of ciliary proteins. The mechanism of how RP2 mutations cause RP is still unclear. In this study, we generated an RP2 knockout zebrafish line using transcription activator-like effector nuclease technology. Progressive retinal degeneration could be observed in the mutant zebrafish. The degeneration of rods' outer segments (OSs) is predominant, followed by the degeneration of cones' OS. These phenotypes are similar to the characteristics of RP2 patients, and also partly consistent with the phenotypes of RP2 knockout mice and morpholino-mediated RP2 knockdown zebrafish. For the first time, we found RP2 deletion leads to decreased protein levels and abnormal retinal localizations of GRK1 and rod transducin subunits (GNAT1 and GNB1) in zebrafish. Furthermore, the distribution of the total farnesylated proteins in zebrafish retina is also affected by RP2 ablation. These molecular alterations observed in the RP2 knockout zebrafish might probably be responsible for the gradual loss of the photoreceptors' OSs. Our work identified the progression of retinal degeneration in RP2 knockout zebrafish, provided a foundation for revealing the pathogenesis of RP caused by RP2 mutations, and would help to develop potential therapeutics against RP in further studies.

Chemical constituents from Sophora tonkinensis and their glucose transporter 4 translocation activities.

Bioassay-guided phytochemical investigation of the EtOAc fraction (ST-EtOAc) from the roots of Sophora tonkinensis resulted in the isolation of a new compound 6aR,11aR-1-hydroxy-4-isoprenyl-maackiain (1), along with 12 known compounds (2-13). The structure of the new compound was established by 1D and 2D NMR, MS data and circular dichroism analysis. Polyprenylated flavonoids 6-9 and 11-13 increased GLUT-4 translocation by the range of 1.35-2.75 folds. Sophoranone (8) exerted the strongest activity with 2.75 folds GLUT-4 translocation enhancement at the concentration of 10µM. This is the first report of the GLUT-4 translocation activity of the plant Sophora tonkinensis.

HSF4 promotes G1/S arrest in human lens epithelial cells by stabilizing p53.

The differentiation from constantly dividing epithelial cells into secondary fiber cells is a key step during lens development. Failure in this process, which requires cell proliferation inhibition and cell cycle exit, causes cataract formation. HSF4 (Heat Shock Transcription Factor 4) gene mutations may lead to both congenital and senile cataract. However, how HSF4 mutations induce cataract formation remains obscure. In this study, we demonstrate that HSF4 can suppress the proliferation of human lens epithelial cells (HLECs) by promoting G1/S arrest in a p53-dependent manner. In contrast, HSF4 with cataract causative mutations fail to cause cell cycle arrest and have no obvious effect on cell proliferation. We further identify that HSF4 recruits p53 in the nucleus and promotes its transcriptional activity, leading to the expression of its target gene p21 in HLECs. HSF4, but not its cataract-causing mutants, stabilizes p53 protein and inhibits its ubiquitin degradation. Our data reveal that HSF4 may work as a switch between lens epithelial cell proliferation and secondary fiber cell differentiation, a process which mainly depends on p53. Through demonstration of this novel downstream pathway of HSF4, our results help uncover the pathogenic mechanisms caused by HSF4 mutations.