A Broad-Spectrum Inhibitor of CRISPR-Cas9.

CRISPR-Cas9 proteins function within bacterial immune systems to target and destroy invasive DNA and have been harnessed as a robust technology for genome editing. Small bacteriophage-encoded anti-CRISPR proteins (Acrs) can inactivate Cas9, providing an efficient off switch for Cas9-based applications. Here, we show that two Acrs, AcrIIC1 and AcrIIC3, inhibit Cas9 by distinct strategies. AcrIIC1 is a broad-spectrum Cas9 inhibitor that prevents DNA cutting by multiple divergent Cas9 orthologs through direct binding to the conserved HNH catalytic domain of Cas9. A crystal structure of an AcrIIC1-Cas9 HNH domain complex shows how AcrIIC1 traps Cas9 in a DNA-bound but catalytically inactive state. By contrast, AcrIIC3 blocks activity of a single Cas9 ortholog and induces Cas9 dimerization while preventing binding to the target DNA. These two orthogonal mechanisms allow for separate control of Cas9 target binding and cleavage and suggest applications to allow DNA binding while preventing DNA cutting by Cas9.

A scoutRNA Is Required for Some Type V CRISPR-Cas Systems.

CRISPR-Cas12c/d proteins share limited homology with Cas12a and Cas9 bacterial CRISPR RNA (crRNA)-guided nucleases used widely for genome editing and DNA detection. However, Cas12c (C2c3)- and Cas12d (CasY)-catalyzed DNA cleavage and genome editing activities have not been directly observed. We show here that a short-complementarity untranslated RNA (scoutRNA), together with crRNA, is required for Cas12d-catalyzed DNA cutting. The scoutRNA differs in secondary structure from previously described tracrRNAs used by CRISPR-Cas9 and some Cas12 enzymes, and in Cas12d-containing systems, scoutRNA includes a conserved five-nucleotide sequence that is essential for activity. In addition to supporting crRNA-directed DNA recognition, biochemical and cell-based experiments establish scoutRNA as an essential cofactor for Cas12c-catalyzed pre-crRNA maturation. These results define scoutRNA as a third type of transcript encoded by a subset of CRISPR-Cas genomic loci and explain how Cas12c/d systems avoid requirements for host factors including ribonuclease III for bacterial RNA-mediated adaptive immunity.

Author Correction: CasX enzymes comprise a distinct family of RNA-guided genome editors.

In this Article, owing to issues with the first 30 nucleotides of the sgRNA, which run in the opposite direction, corrections have been made to the Protein Data Bank (PDB) accessions in the 'Data availability' section, and this also affects Figs. 3, 4, Extended Data Fig. 6, Supplementary Table 1 and Supplementary Video 1. The original Article has been corrected online. See the accompanying Amendment for further details.

CasX enzymes comprise a distinct family of RNA-guided genome editors.

The RNA-guided CRISPR-associated (Cas) proteins Cas9 and Cas12a provide adaptive immunity against invading nucleic acids, and function as powerful tools for genome editing in a wide range of organisms. Here we reveal the underlying mechanisms of a third, fundamentally distinct RNA-guided genome-editing platform named CRISPR-CasX, which uses unique structures for programmable double-stranded DNA binding and cleavage. Biochemical and in vivo data demonstrate that CasX is active for Escherichia coli and human genome modification. Eight cryo-electron microscopy structures of CasX in different states of assembly with its guide RNA and double-stranded DNA substrates reveal an extensive RNA scaffold and a domain required for DNA unwinding. These data demonstrate how CasX activity arose through convergent evolution to establish an enzyme family that is functionally separate from both Cas9 and Cas12a.

Improved genome editing by an engineered CRISPR-Cas12a.

CRISPR-Cas12a is an RNA-guided, programmable genome editing enzyme found within bacterial adaptive immune pathways. Unlike CRISPR-Cas9, Cas12a uses only a single catalytic site to both cleave target double-stranded DNA (dsDNA) (cis-activity) and indiscriminately degrade single-stranded DNA (ssDNA) (trans-activity). To investigate how the relative potency of cis- versus trans-DNase activity affects Cas12a-mediated genome editing, we first used structure-guided engineering to generate variants of Lachnospiraceae bacterium Cas12a that selectively disrupt trans-activity. The resulting engineered mutant with the biggest differential between cis- and trans-DNase activity in vitro showed minimal genome editing activity in human cells, motivating a second set of experiments using directed evolution to generate additional mutants with robust genome editing activity. Notably, these engineered and evolved mutants had enhanced ability to induce homology-directed repair (HDR) editing by 2-18-fold compared to wild-type Cas12a when using HDR donors containing mismatches with crRNA at the PAM-distal region. Finally, a site-specific reversion mutation produced improved Cas12a (iCas12a) variants with superior genome editing efficiency at genomic sites that are difficult to edit using wild-type Cas12a. This strategy establishes a pipeline for creating improved genome editing tools by combining structural insights with randomization and selection. The available structures of other CRISPR-Cas enzymes will enable this strategy to be applied to improve the efficacy of other genome-editing proteins.

Single-Stranded DNA Cleavage by Divergent CRISPR-Cas9 Enzymes.

Double-stranded DNA (dsDNA) cleavage by Cas9 is a hallmark of type II CRISPR-Cas immune systems. Cas9-guide RNA complexes recognize 20-base-pair sequences in DNA and generate a site-specific double-strand break, a robust activity harnessed for genome editing. DNA recognition by all studied Cas9 enzymes requires a protospacer adjacent motif (PAM) next to the target site. We show that Cas9 enzymes from evolutionarily divergent bacteria can recognize and cleave single-stranded DNA (ssDNA) by an RNA-guided, PAM-independent recognition mechanism. Comparative analysis shows that in contrast to the type II-A S. pyogenes Cas9 that is widely used for genome engineering, the smaller type II-C Cas9 proteins have limited dsDNA binding and unwinding activity and promiscuous guide RNA specificity. These results indicate that inefficiency of type II-C Cas9 enzymes for genome editing results from a limited ability to cleave dsDNA and suggest that ssDNA cleavage was an ancestral function of the Cas9 enzyme family.

Transcription factors, cap 'n' collar isoform C regulates the expression of CYP450 genes involving in insecticides susceptibility in Locusta migratoria.

BACKGROUND: The cap 'n' collar (Cnc) belongs to the Basic Leucine Zipper (bZIP) transcription factor super family. Cap 'n' collar isoform C (CncC) is highly conserved in the animal kingdom. CncC contributes to the regulation of growth, development, and aging and takes part in the maintenance of homeostasis and the defense against endogenous and environmental stress. Insect CncC participates in the regulation of various kinds of stress-responsive genes and is involved in the development of insecticide resistance. RESULTS: In this study, one full-length CncC sequence of Locusta migratoria was identified and characterized. Upon RNAi silencing of LmCncC, insecticide bioassays showed that LmCncC played an essential role in deltamethrin and imidacloprid susceptibility. To fully investigate the downstream genes regulated by LmCncC and further identify the LmCncC-regulated genes involved in deltamethrin and imidacloprid susceptibility, a comparative transcriptome was constructed. Thirty-five up-regulated genes and 73 down-regulated genes were screened from dsLmCncC-knockdown individuals. We selected 22 LmCncC-regulated genes and verified their gene expression levels using RT-qPCR. Finally, six LmCYP450 genes belonging to the CYP6 family were selected as candidate detoxification genes, and LmCYP6FD1 and LmCYP6FE1 were further validated as detoxification genes of insecticides via RNAi, insecticide bioassays, and metabolite identification. CONCLUSIONS: Our data suggest that the locust CncC gene is associated with deltamethrin and imidacloprid susceptibility via the regulation of LmCYP6FD1 and LmCYP6FE1, respectively.

Nontoxic nanopore electroporation for effective intracellular delivery of biological macromolecules.

We present a simple nanopore-electroporation (NanoEP) platform for delivery of nucleic acids, functional protein, and Cas9 single-guide RNA ribonucleoproteins into both adherent and suspension cells with up to 80% delivery efficiency and >95% cell viability. Low-voltage electric pulses permeabilize a small area of cell membrane as a cell comes into close contact with the nanopores. The biomolecule cargo is then electrophoretically drawn into the cells through the nanopores. In addition to high-performance delivery with low cell toxicity, the NanoEP system does not require specialized buffers, expensive materials, complicated fabrication processes, or cell manipulation; it simply consists of a generic nanopore-embedded water-filter membrane and a low-voltage square-wave generator. Ultimately, the NanoEP platform offers an effective and flexible method for universal intracellular delivery.

DEAD-Box RNA Helicase DDX47 Maintains Midgut Homeostasis in Locusta migratoria.

Tissue homeostasis is critical for maintaining organ shape, size, and function. The condition is regulated by the balance between the generation of new cells and the loss of senescent cells, and it involves many factors and mechanisms. The midgut, an important part of the intestinal tract, is responsible for digestion and nutrient absorption in insects. LmDDX47, the ortholog of DEAD-box helicase 47 from Locusta migratoria, is indispensable for sustaining a normal midgut in the nymphs. However, the underlying cellular and molecular mechanisms remain to be elucidated. In this study, LmDDX47 knockdown resulted in atrophy of the midgut and gastric cecum in both nymph and adult locusts. After LmDDX47 knockdown, the number of regenerative and columnar cells in the midgut was significantly reduced, and cell death was induced in columnar tissue. LmDDX47 was localized to the nucleolus; this was consistent with the reduction in 18S rRNA synthesis in the LmDDX47 knockdown group. In addition, the acetylation and crotonylation levels of midgut proteins were significantly increased. Therefore, LmDDX47 could be a key regulator of midgut homeostasis, regulating 18S rRNA synthesis as well as protein acetylation and crotonylation in the migratory locust.

The RNA helicase DDX3 is required for ovarian development and oocyte maturation in Locusta migratoria.

DDX3 represents a well-defined subfamily of DEAD-box RNA helicase and exerts multiple functions in RNA metabolism, cell cycle, tumorigenesis, signal pathway, and fertility. Our previous study has shown that LmDDX3, the ortholog of DDX3 in Locusta migratoria, is ubiquitously expressed, and with a high abundance in testis and ovary. Knockdown of LmDDX3 results in a lethal phenotype in nymph, but it still remains unclear for its role in reproductive process. In this study, we therefore characterized LmDDX3 expression in female adult locust and analyzed its function in oocyte development. LmDDX3 was expressed in all tissues examined with significant more transcripts in ovary and hindgut. In ovary, a strong expression level was detected at the day just after adult eclosion, and a dramatic reduction then occurred during the oocyte development. LmDDX3 RNAi led to a reduced vitellogenin (Vg) expression in fat body via partially at least, the JH signaling pathway, and caused an upregulation of vitellogenin receptor (VgR) in ovary, and thus blocked the ovarian development and oocyte maturation. Sequence and phylogenetic analysis indicated that LmDDX3 was closely related to termite DDX3. Taken together, these data reveal a critical role for LmDDX3 in regulating the transcription of Vg and VgR, two major factors in vitellogenesis that is a key process required for ovary development and oocyte maturation in locust, and contribute thereof a new putative target for locust biological control.

Dietary pattern changes in Fukushima residents after the Great East Japan Earthquake: the Fukushima Health Management Survey 2011-2013.

OBJECTIVE: Dietary patterns more closely resemble actual eating behaviours because multiple food groups, not a single food group or nutrient, are considered. The present study aimed to identify and assess changes of dietary patterns in Fukushima residents. DESIGN: Dietary data were collected using a short-form FFQ in annual Fukushima Health Management Survey between 2011 and 2013 after the Great East Japan Earthquake. Year- and sex-specific dietary patterns were determined by the principal component analysis. SETTING: Evacuation and nonevacuation zones in Fukushima, Japan. PARTICIPANTS: Eligible participants aged ≥16 years answered the FFQ (n 67 358 in 2011, n 48 377 in 2012 and n 40 742 in 2013). RESULTS: Three identified dietary patterns were assessed similarly in men and women and among years: typical, juice and meat. In total participants, the Spearman's correlation coefficients between two survey years were 0·70-0·74 for the typical, 0·58-0·66 for the juice and 0·50-0·54 for the meat pattern scores. Adjusted for sociodemographic factors, evacuees had lower typical pattern scores, higher juice pattern scores and the same meat pattern scores compared with non-evacuees. The means of typical pattern scores in evacuees and it of juice pattern scores in non-evacuees continued declining over years. Similar profiles of dietary patterns and trends of pattern scores were observed in participants (n 22 805) who had provided three dietary assessments. CONCLUSIONS: Changes of dietary patterns have been observed between 2011 and 2013. Careful investigation of those with low intake of typical pattern foods and promotion of them, particularly in evacuees, are needed.

Short-Term Effects of Early Postoperative Celecoxib Administration for Pain, Sleep Quality, and Range of Motion After Total Knee Arthroplasty: A Randomized Controlled Trial.

BACKGROUND: We hypothesized that early postoperative administration of celecoxib would reduce pain scores and improve sleep quality and active range of motion after total knee arthroplasty (TKA) under general anesthesia. METHODS: Patients in the celecoxib group received 400 mg of celecoxib 2 hours after TKA, followed 6 hours later by 200 mg of celecoxib. Patients in the control group received 400 mg of celecoxib the second day after surgery. Patients in both group had access to patient-controlled analgesia fentanyl. The primary outcome measure was the patient-reported visual analog scale (VAS) pain score the second day after TKA. The secondary outcome measure was sleep quality (days 1, 2, and 7 postoperatively). Active knee joint range of motion was assessed on days 2 and 7 postoperatively, and VAS pain scores were evaluated on postoperative days 1 to 7. Total fentanyl consumption was also assessed. RESULTS: Compared to the control group, the celecoxib group had significantly lower median VAS pain scores on postoperative days 1 and 2, significantly less nocturnal awakening (in minutes) and frequency of body motion, and better sleep efficacy on postoperative day 1. The celecoxib group also had a significantly better median flexion angle (°) on postoperative days 2 and 7, and lower cumulative fentanyl consumption. CONCLUSION: Early administration of celecoxib after TKA was associated with significantly reduced early VAS pain scores and improved sleep quality and active knee flexion angles. Thus, the early administration of celecoxib after TKA under general anesthesia may reduce pain and improve sleep quality and functional recovery. LEVELS OF EVIDENCE: Level II, therapeutic study. TRIAL REGISTRATION: UMIN-CTR 000014624 (July 23, 2014).

A second intracellular copper/zinc superoxide dismutase and a manganese superoxide dismutase in Oxya chinensis: Molecular and biochemical characteristics and roles in chlorpyrifos stress.

A second intracellular copper/zinc superoxide dismutase (icCuZnSOD2) and manganese SOD (MnSOD) were cloned and characterized in Oxya chinensis. The open reading frame (ORF) of OcicCuZnSOD2 and OcMnSOD are 462 and 672 bp encoding 153 and 223 amino acids, respectively. OcicCuZnSOD2 contains two signature sequences, one potential N-glycosylation site, and seven copper/zinc binding sites. OcMnSOD includes a mitochondria targeting sequence of 7 amino acids at N-terminal, one signature sequence, two N-glycosylation sites, and four manganese binding sites. The secondary structure and homology model of OcicCuZnSOD2 include nine ß sheets, two Greek-key motifs, and one electrostatic loop. OcMnSOD contains nine α-helices and three ß-sheets. Phylogenetic analysis shows that OcMnSOD is evolutionarily conserved while OcicCuZnSOD2 may be gene duplication and is paralogous to OcicCuZnSOD1. OcMnSOD expressed widely in all tissues and developmental stages. OcicCuZnSOD2 showed testis-specific expression and expressed highest in the 5th-instar nymph and the adult. The optimum temperatures and pH values of the recombinant OcicCuZnSOD2 and OcMnSOD were 40 °C and 8.0. They were stable at 25-55 °C and at pH 5.0-12.0 and pH 6.0-12.0, respectively. The activity and mRNA expression of each OcSOD were assayed after chlorpyrifos treatments. Total SOD and CuZnSOD activities first increased then declined under chlorpyrifos stress. Chlorpyrifos induced the mRNA expression and activity of OcMnSOD as a dose-dependent manner and inhibited OcicCuZnSOD2 transcription. The role of each OcSOD gene in chlorpyrifos stress was investigated using RNAi and disc diffusion assay with Escherichia coli overexpressing OcSOD proteins. Silencing of OcMnSOD significantly increased ROS content in chlorpyrifos-exposed grasshoppers. Disc diffusion assay showed that the plates with E. coli overexpressing OcMnSOD had the smaller inhibition zones around the chlorpyrifos-soaked filter discs. These results implied that OcMnSOD played a significant role in defense chlorpyrifos-induced oxidative stress.

Multiple Argonaute family genes contribute to the siRNA-mediated RNAi pathway in Locusta migratoria.

Argonautes (Ago) are important core proteins in RNA interference (RNAi) pathways of eukaryotic cells. Generally, it is thought that Ago1, Ago2 and Ago3 are involved in the miRNA (microRNA), siRNA (small interfering RNA) and piRNA (Piwi-interacting RNA)-mediated RNAi pathways, respectively. As a main component of the RNA-induced silencing complex (RISC), Ago2 plays an indispensable role in using siRNA to recognize and cut target messenger RNAs resulting in suppression of transcript levels, but the contributions of Ago1 and Ago3 to the siRNA-mediated RNAi pathway remain to be explored in many insect species. In this study, we investigated the contributions of four Ago genes (named LmAgo1, LmAgo2a and LmAgo2b and LmAgo3) to RNAi efficiency in Locusta migratoria by using both in vivo and in vitro experiments. Our results showed that suppression of each of the Ago genes significantly impaired RNAi efficiency when targeting Lmß-tubulin transcripts, resulting in recovery of 48, 43.3, 61.4 or 26% of Lmß-tubulin transcripts following RNAi-mediated suppression of LmAgo1, LmAgo2a, LmAgo2b, and LmAgo3, respectively. Furthermore, overexpression of LmAgo1, LmAgo2a, LmAgo2b, or LmAgo3 in a PAc5.1-V5/HisB vector and co-transfection with psicheck2 fluorescence vector in S2 cells reduced luciferase fluorescence by 38.3, 58.9, 53.3 or 55.6%, respectively. Taken together, our results showed that LmAgo1, LmAgo2a, LmAgo2b, and LmAgo3 each make significant contributions to RNAi efficiency in L. migratoria and suggest that the involvement of all four enzymes could be one of the major factors supporting robust RNAi responses observed in this species.

Knockdown of LmCYP303A1 alters cuticular hydrocarbon profiles and increases the susceptibility to desiccation and insecticides in Locusta migratoria.

Cytochrome P450 monooxygenases (CYPs) serve many functions in insects, from the regulation of development to xenobiotic detoxification. Several conserved CYPs have been shown to play a role in insect growth and development. CYP303A1 is a highly conserved CYP with a single ortholog in most insects, but its underlying molecular characteristics and specific physiological functions remain poorly understood. In Drosophila melanogaster and Locusta migratoria, CYP303A1 is indispensable for eclosion to adult. Here, we report additional functions of the locust gene LmCYP303A1 in nymphal molts, cuticular lipid deposition and insecticide penetration. RT-qPCR revealed that LmCYP303A1 had a high expression level before ecdysis and was highly expressed in integument, wing pads, foregut and hindgut. Suppression of LmCYP303A1 expression by RNA interference (RNAi) caused a lethal phenotype with molting defect from nymph to nymph. In addition, LmCYP303A1 RNAi resulted in locusts being more susceptible to desiccation and to insecticide toxicity. Furthermore, knockdown of LmCYP303A1 efficiently suppressed the transcript level of key genes (ELO7, FAR15 and CYP4G102) responsible for cuticular hydrocarbon (CHC) synthesis, which led to a decrease in some CHC levels. Taken together, our results suggest that one of the functions of LmCYP303A1 is to regulate the biosynthesis of CHC, which plays critical roles in protecting locusts from water loss and insecticide penetration.

siRNA repositioning for guide strand selection by human Dicer complexes.

The human ribonuclease Dicer and its double-stranded RNA (dsRNA)-binding protein (dsRBP) partners TRBP and PACT play important roles in the biogenesis of regulatory RNAs. Following dicing, one dsRNA product strand is preferentially assembled into an RNA-induced silencing complex (RISC). The mechanism of strand selection in humans and the possible role of Dicer in this process remain unclear. Here we demonstrate that dsRNAs undergo significant repositioning within Dicer complexes following dicing. This repositioning enables directional binding of RNA duplexes, thereby biasing their orientation for guide strand selection according to the thermodynamic properties of the helix. Our findings indicate that Dicer is itself capable of sensing siRNA thermodynamic asymmetry regardless of the dsRBP to which it is bound. These results support a model in which Dicer employs two distinct RNA-binding sites-one for dsRNA processing and the other for sensing of siRNA thermodynamic asymmetry-during RISC loading in humans.

Antioxidant defenses at enzymatic and transcriptional levels in response to acute lead administration in Oxya chinensis.

Lead (Pb) is known to be toxic to many organisms. Oxidative stress is a major mechanism of its toxicity. This research aims to investigate the effects of Pb on hydrogen peroxide (H2O2) and malonedialdehyde (MDA) contents, activities and mRNA levels of antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx)) after Oxya chinensis were acutely treated with lead acetate for 24 h. The results showed that the LD50-24 h value of lead acetate to O. chinensis was 1541.89 (1431.19-1655.77) µg g-1 body H2O2 and MDA contents were elevated after Pb administration, which suggested that Pb induced the overproduction of ROS and caused oxidative stress. SOD activities were significantly inhibited 40.42% of the control by 280 µg µL-1 Pb. CAT activities were increased while GPx activities had no significant changes. Different types of antioxidant-related genes had various responses to Pb stress. The transcriptions of icCuZnSOD2 and ecCuZnSOD2 were significantly inhibited by different concentrations of Pb. MnSOD mRNA levels showed the concentration-dependent rise with the Pb concentrations increase. The expressions of ecCuZnSOD1, CAT1, and GPx were significantly up-regulated while the transcriptions of icCuZnSOD1 and CAT2 had no significant changes. Alteration of activities and mRNA expressions of antioxidant enzymes implied that Pb-induced antioxidant defenses were related to modifications at enzymatic and transcriptional levels. The profiles of antioxidant enzymes and H2O2 and MDA contents and relationships among the parameters indicated that the cooperation of multiple antioxidants rather than a single factor might be responsible for the antioxidant defenses against Pb stress.

Metabolism of selected model substrates and insecticides by recombinant CYP6FD encoded by its gene predominately expressed in the brain of Locusta migratoria.

The migratory locust, Locusta migartoria, is a major agricultural insect pest and its resistance to insecticides is becoming more prevalent. Cytochrome P450 monooxygenases (CYPs) are important enzymes for biotransformations of various endogenous and xenobiotic substances. These enzymes play a major role in developing insecticide resistance in many insect species. In this study, we heterologously co-expressed a CYP enzyme (CYP6FD1) and cytochrome P450 reductase (CPR) from L. migartoria in Sf9 insect cells. The recombinant enzymes were assayed for metabolic activity towards six selected model substrates (luciferin-H, luciferin-Me, luciferin-Be, luciferin-PFBE, luciferin-CEE and 7-ethoxycoumarin), and four selected insecticides (deltamethrin, chlorpyrifos, carbaryl and methoprene). Recombinant CYP6FD1 showed activity towards 7-ethoxycoumarin and luciferin-Me, but no detectable activity towards the other luciferin derivatives. Furthermore, the enzyme efficiently oxidized deltamethrin to hydroxydeltamethrin through an aromatic hydroxylation in a time-dependent manner. However, the enzyme did not show any detectable activity towards the other three insecticides. Our results provide direct evidence that CYP6FD1 is capable of metabolizing deltamethrin. This work is a step towards a more complete characterization of the catalytic capabilities of CYP6FD1 and other xenobiotic metabolizing CYP enzymes in L. migratoria.

Brazilin induces T24 cell death through c-Fos and GADD45ß independently regulated genes and pathways.

Purified Brazilin from Sappan wood extract has been reported with significant antitumor effect, especially on human T24 cells and bladder cancer mouse models. Brazilin can significantly induce expression of c-Fos and GADD45ß and transfection expression of c-Fos and GADD45ß in T24 cells can induce significant cell morphology changes, reduced viability and cell death, while transfection of siRNA-c-Fos and siRNA-GADD45ß can reverse the induced cell death. Co-transfection of both c-Fos and GADD45ß into T24 cells resulted in a significantly additive effect when compared to single transfection with only c-Fos or GADD45ß. Meanwhile, transfection of interfering siRNA-c-Fos or siRNA-GADD45ß can partially rescue the cell viability and siRNA co-transfection showed increased rescue rate. The transfection expression and interference with pcDNA3.1-c-Fos/siRNA-c-Fos or pcDNA3.1-GADD45ß/siRNA-GADD45ß did not affect each other's expression. Moreover, analysis of c-Fos and GADD45ß regulated genes and signal pathways showed that no common regulated genes or pathways were present. All the results indicated that c-Fos and GADD45ß mediate independent Brazilin-inducible genes and pathways. © 2018 IUBMB Life, 70(11):1101-1110, 2018.

Dietary Antioxidant Micronutrients and All-Cause Mortality: The Japan Collaborative Cohort Study for Evaluation of Cancer Risk.

BACKGROUND: Oxidative stress, the imbalance between pro- and antioxidants, has been implicated in the etiology and pathophysiology of the incidence and mortality of many diseases. We aim to investigate the relations of dietary intakes of vitamin C and E and main carotenoids with all-cause mortality in Japanese men and women. METHODS: The Japan Collaborative Cohort Study for Evaluation of Cancer Risk had 22,795 men and 35,539 women, aged 40-79 years at baseline (1988-1990), who completed a valid food frequency questionnaire and were followed up to the end of 2009. RESULTS: There were 6,179 deaths in men and 5,355 deaths in women during the median follow-up of 18.9 years for men and 19.4 years for women. Multivariate hazard ratios for the highest versus lowest quintile intakes in women were 0.83 (95% confidence interval [CI], 0.76-0.90; P for trend < 0.0001) for vitamin C, 0.85 (95% CI, 0.78-0.93; P for trend < 0.0001) for vitamin E, 0.88 (95% CI, 0.81-0.96; P for trend = 0.0006) for ß-carotene, and 0.90 (95% CI, 0.82-0.98; P for trend = 0.0002) for ß-cryptoxanthin. The joint effect of any two of these highly correlated micronutrients showed significant 12-17% reductions in risk in the high-intake group compared with the low-intake group in women. These significant associations were also observed in the highest quintile intakes of vitamin C, vitamin E, and ß-carotene in female non-smokers but were not observed in female smokers, male smokers, and non-smokers. CONCLUSIONS: Higher dietary intakes of antioxidant vitamins may reduce the risk of all-cause mortality in middle-aged Japanese women, especially female non-smokers.