A scanning-to-incision switch in TFIIH-XPG induced by DNA damage licenses nucleotide excision repair.

Nucleotide excision repair (NER) is critical for removing bulky DNA base lesions and avoiding diseases. NER couples lesion recognition by XPC to strand separation by XPB and XPD ATPases, followed by lesion excision by XPF and XPG nucleases. Here, we describe key regulatory mechanisms and roles of XPG for and beyond its cleavage activity. Strikingly, by combing single-molecule imaging and bulk cleavage assays, we found that XPG binding to the 7-subunit TFIIH core (coreTFIIH) stimulates coreTFIIH-dependent double-strand (ds)DNA unwinding 10-fold, and XPG-dependent DNA cleavage by up to 700-fold. Simultaneous monitoring of rates for coreTFIIH single-stranded (ss)DNA translocation and dsDNA unwinding showed XPG acts by switching ssDNA translocation to dsDNA unwinding as a likely committed step. Pertinent to the NER pathway regulation, XPG incision activity is suppressed during coreTFIIH translocation on DNA but is licensed when coreTFIIH stalls at the lesion or when ATP hydrolysis is blocked. Moreover, ≥15 nucleotides of 5'-ssDNA is a prerequisite for efficient translocation and incision. Our results unveil a paired coordination mechanism in which key lesion scanning and DNA incision steps are sequentially coordinated, and damaged patch removal is only licensed after generation of ≥15 nucleotides of 5'-ssDNA, ensuring the correct ssDNA bubble size before cleavage.

Nucleotide excision repair (NER) removes bulky DNA lesions and is thereby crucial in maintaining transcription and genomic integrity. Here, the authors show a dual function for the XPG nuclease that is critical for finding and excising the damage. During the separation of the damage-containing strand from the undamaged strand, XPG stimulates TFIIH dependent dsDNA unwinding 10 fold. In return, when TFIIH stalls at the damage it stimulates XPG nuclease activity 700 fold. Remarkably, this mutually exclusive coordination requires a bubble longer than 15 nucleotides. This study addressees why a bubble of a certain size is needed to facilitate NER and why XPG is recruited at the beginning of NER when its endonucleolytic activity is required at the very end.

Bt corn and cotton planting may benefit peanut growers by reducing aflatoxin risk.

Decades of studies have shown that Bt corn, by reducing insect damage, has lower levels of mycotoxins (fungal toxins), such as aflatoxin and fumonisin, than conventional corn. We used crop insurance data to infer that this benefit from Bt crops extends to reducing aflatoxin risk in peanuts: a non-Bt crop. In consequence, we suggest that any benefit-cost assessment of how transgenic Bt crops affect food safety should not be limited to assessing those crops alone; because the insect pest control offered by Bt crops affects the food safety profile of other crops grown nearby. Specifically, we found that higher Bt corn and Bt cotton planting rates in peanut-growing areas of the United States were associated with lower aflatoxin risk in peanuts as measured by aflatoxin-related insurance claims filed by peanut growers. Drought-related insurance claims were also lower: possibly due to Bt crops' suppression of insects that would otherwise feed on roots, rendering peanut plants more vulnerable to drought. These findings have implications for countries worldwide where policies allow Bt cotton but not Bt food crops to be grown: simply planting a Bt crop may reduce aflatoxin and drought stress in nearby food crops, resulting in a safer food supply through an inter-crop "halo effect."

Modulation of Enzyme-Catalyzed Synthesis of Prostaglandins by Components Contained in Kidney Microsomal Preparations.

In the kidney, prostaglandins formed by cyclooxygenase 1 and 2 (COX-1 and COX-2) play an important role in regulating renal blood flow. In the present study, we report our observations regarding a unique modulatory effect of renal microsomal preparation on COX-1/2-mediated formation of major prostaglandin (PG) products in vitro. We found that microsomes prepared from pig and rat kidneys had a dual stimulatory-inhibitory effect on the formation of certain PG products catalyzed by COX-1 and COX-2. At lower concentrations, kidney microsomes stimulated the formation of certain PG products, whereas at higher concentrations, their presence inhibited the formation. Presence of kidney microsomes consistently increased the Km values of the COX-1/2-mediated reactions, while the Vmax might be increased or decreased depending on stimulation or inhibition observed. Experimental evidence was presented to show that a protein component present in the pig kidney microsomes was primarily responsible for the activation of the enzyme-catalyzed arachidonic acid metabolism leading to the formation of certain PG products.

Immune Modulatory Activities of Arginyl-Fructose (AF) and AF-Enriched Natural Products in In-Vitro and In-Vivo Animal Models.

The immune system plays an important role in maintaining body homeostasis. Recent studies on the immune-enhancing effects of ginseng saponins have revealed more diverse mechanisms of action. Maillard reaction that occurs during the manufacturing processes of red ginseng produces a large amount of Amadori rearrangement compounds (ARCs), such as arginyl-fructose (AF). The antioxidant and anti-hyperglycemic effects of AF have been reported. However, the possible immune enhancing effects of non-saponin ginseng compounds, such as AF, have not been investigated. In this study the effects of AF and AF-enriched natural product (Ginofos, GF) on proliferation of normal mouse splenocytes were evaluated in vitro and male BALB/c mice models. The proliferation of splenocytes treated with mitogens (concanavalin A, lipopolysaccharide) were further increased by addition of AF (p < 0.01) or GF (p < 0.01), in a dose dependent manner. After the 10 days of oral administration of compounds, changes in weights of spleen and thymus, serum immunoglobulin, and expression of cytokines were measured as biomarkers of immune-enhancing potential in male BALB/c mice model. The AF or GF treated groups had higher weights of the thymus (0.94 ± 0.25 and 0.86 ± 0.18, p < 0.05, respectively) than that of cyclophosphamide treated group (0.59 ± 0.18). This result indicates that AF or AF-enriched extract (GF) increased humoral immunity against CY-induced immunosuppression. In addition, immunoglobulin contents and expression of cytokines including IgM (p < 0.01), IgG (p < 0.05), IL-2 (p < 0.01), IL-4 (p < 0.01), IL-6 (p < 0.01), and IFN-γ (p < 0.05) were also significantly increased by supplementation of AF or GF. These results indicate that AF has immune enhancing effects by activation of adaptive immunity via increase of expression of immunoglobulins and cytokines such as IgM, IgG, IL-2, IL-4, IL-6 and thereby proliferating the weight of thymus. Our findings provide a pharmacological rationale for AF-enriched natural products such as ginseng and red ginseng that can possibly have immune-enhancement potential and should be further evaluated.

Lack of Cell Proliferative and Tumorigenic Effects of 4-Hydroxyestradiol in the Anterior Pituitary of Rats: Role of Ultrarapid O-Methylation Catalyzed by Pituitary Membrane-Bound Catechol-O-Methyltransferase.

In animal models, estrogens are complete carcinogens in certain target sites. 4-Hydroxyestradiol (4-OH-E2), an endogenous metabolite of 17ß-estradiol (E2), is known to have prominent estrogenic activity plus potential genotoxicity and mutagenicity. We report here our finding that 4-OH-E2 does not induce pituitary tumors in ACI female rats, whereas E2 produces 100% pituitary tumor incidence. To probe the mechanism, we conducted a short-term animal experiment to compare the proliferative effect of 4-OH-E2 in several organs. We found that, whereas 4-OH-E2 had little ability to stimulate pituitary cell proliferation in ovariectomized female rats, it strongly stimulates cell proliferation in certain brain regions of these animals. Further, when we used in vitro cultured rat pituitary tumor cells as models, we found that 4-OH-E2 has similar efficacy as E2 in stimulating cell proliferation, but its potency is approximately 3 orders of magnitude lower than that of E2. Moreover, we found that the pituitary tumor cells have the ability to selectively metabolize 4-OH-E2 (but not E2) with ultrahigh efficiency. Additional analysis revealed that the rat pituitary expresses a membrane-bound catechol-O-methyltransferase that has an ultralow Km value (in nM range) for catechol estrogens. On the basis of these observations, it is concluded that rapid metabolic disposition of 4-OH-E2 through enzymatic O-methylation in rat anterior pituitary cells largely contributes to its apparent lack of cell proliferative and tumorigenic effects in this target site.

Sorafenib for 9,923 Patients with Hepatocellular Carcinoma: An Analysis from National Health Insurance Claim Data in South Korea.

Background/Aims: Sorafenib is the standard of care in the management of advanced hepatocellular carcinoma (HCC). The purpose of this study was to investigate the characteristics, treatment patterns and outcomes of sorafenib among HCC patients in South Korea. Methods: This population-based retrospective, single-arm, observational study used the Korean National Health Insurance database to identify patients with HCC who received sorafenib between July 1, 2008, and December 31, 2014. A total of 9,923 patients were recruited in this study. Results: Among 9,923 patients, 6,669 patients (68.2%) received loco-regional therapy prior to sorafenib, and 1,565 patients (15.8%) received combination therapy with concomitant sorafenib; 2,591 patients (26.1%) received rescue therapy after sorafenib, and transarterial chemoembolization was the most common modality applied in 1,498 patients (15.1%). A total of 3,591 patients underwent rescue therapy after sorafenib, and the median overall survival was 14.5 months compared to 4.6 months in 7,332 patients who received supportive care after sorafenib. The mean duration of sorafenib administration in all patients was 105.7 days; 7,023 patients (70.8%) received an initial dose of 600 to 800 mg. The longest survival was shown in patients who received the recommended dose of 800 mg, subsequently reduced to 400 mg (15.0 months). The second longest survival was demonstrated in patients with a starting dose of 800 mg, followed by a dose reduction to 400-600 mg (9.6 months). Conclusions: Real-life data show that the efficacy of sorafenib seems similar to that observed in clinical trials, suggesting that appropriate subsequent therapy after sorafenib might prolong patient survival.

Dynamic conformational switching underlies TFIIH function in transcription and DNA repair and impacts genetic diseases.

Transcription factor IIH (TFIIH) is a protein assembly essential for transcription initiation and nucleotide excision repair (NER). Yet, understanding of the conformational switching underpinning these diverse TFIIH functions remains fragmentary. TFIIH mechanisms critically depend on two translocase subunits, XPB and XPD. To unravel their functions and regulation, we build cryo-EM based TFIIH models in transcription- and NER-competent states. Using simulations and graph-theoretical analysis methods, we reveal TFIIH's global motions, define TFIIH partitioning into dynamic communities and show how TFIIH reshapes itself and self-regulates depending on functional context. Our study uncovers an internal regulatory mechanism that switches XPB and XPD activities making them mutually exclusive between NER and transcription initiation. By sequentially coordinating the XPB and XPD DNA-unwinding activities, the switch ensures precise DNA incision in NER. Mapping TFIIH disease mutations onto network models reveals clustering into distinct mechanistic classes, affecting translocase functions, protein interactions and interface dynamics.

Mechanism of Rad26-assisted rescue of stalled RNA polymerase II in transcription-coupled repair.

Transcription-coupled repair is essential for the removal of DNA lesions from the transcribed genome. The pathway is initiated by CSB protein binding to stalled RNA polymerase II. Mutations impairing CSB function cause severe genetic disease. Yet, the ATP-dependent mechanism by which CSB powers RNA polymerase to bypass certain lesions while triggering excision of others is incompletely understood. Here we build structural models of RNA polymerase II bound to the yeast CSB ortholog Rad26 in nucleotide-free and bound states. This enables simulations and graph-theoretical analyses to define partitioning of this complex into dynamic communities and delineate how its structural elements function together to remodel DNA. We identify an allosteric pathway coupling motions of the Rad26 ATPase modules to changes in RNA polymerase and DNA to unveil a structural mechanism for CSB-assisted progression past less bulky lesions. Our models allow functional interpretation of the effects of Cockayne syndrome disease mutations.

Author Correction: The Impact of Bt Corn on Aflatoxin-Related Insurance Claims in the United States.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The Impact of Bt Corn on Aflatoxin-Related Insurance Claims in the United States.

Previous field studies have reached no collective consensus on whether Bt corn, the most commonly planted transgenic crop worldwide, has significantly lower aflatoxin levels than non-Bt isolines. Aflatoxin, a mycotoxin contaminating corn and other commodities, causes liver cancer in humans and can pose severe economic losses to farmers. We found that from 2001-2016, a significant inverse correlation existed between Bt corn planting and aflatoxin-related insurance claims in the United States, when controlling for temperature and drought. Estimated benefits of aflatoxin reduction resulting from Bt corn planting are about $120 million to $167 million per year over 16 states on average. These results suggest that Bt corn use is an important strategy in reducing aflatoxin risk, with corresponding economic benefits. If the same principles hold true in other world regions, then Bt corn hybrids adapted to diverse agronomic regions may have a role in reducing aflatoxin in areas prone to high aflatoxin contamination, and where corn is a dietary staple.

Naturally-occurring estradiol-17beta-fatty acid esters, but not estradiol-17beta, preferentially induce mammary tumorigenesis in female rats: implications for an important role in human breast cancer.

Because mammary glands are surrounded by adipose tissues, we hypothesize that the ultra-lipophilic endogenous estrogen-17beta-fatty acid esters may have preferential hormonal and carcinogenic effects in mammary tissues compared to other target organs (such as the uterus and pituitary). This hypothesis is tested in the present study. We found that all 46 rats implanted with an estradiol-17beta pellet developed large pituitary tumors (average weight=251+/-103 mg) and had to be terminated early, but only 48% of them developed mammary tumors. In addition, approximately one-fourth of them developed a huge uterus. In the 26 animals implanted with a mixture containing estradiol-17beta-stearate and estradiol-17beta-palmitate (two representative estradiol-17beta-fatty acid esters) or in the 29 animals implanted with estradiol-17beta-stearate alone (in the same molar dose as estradiol-17beta), 73% and 79%, respectively, of them developed mammary tumors, whereas only 3 or 2 animals, respectively, had to be terminated early due to the presence of a large pituitary tumor. Both tumorous and normal mammary tissues contained much higher levels of estrogen esterase than other tissues, which catalyzes the releases of bioactive estrogens from their fatty acid esters. In conclusion, while estradiol-17beta is much stronger in inducing pituitary tumor (100% incidence) than mammary tumor, estradiol-17beta-fatty acid esters have a higher efficacy than estradiol-17beta in inducing mammary tumor and yet it only has little ability to induce uterine out-growth and pituitary tumorigenesis. This study establishes the endogenous estrogen-17beta-fatty acid esters as preferential inducers of mammary tumorigenesis.

Biochemical and molecular modeling studies of the O-methylation of various endogenous and exogenous catechol substrates catalyzed by recombinant human soluble and membrane-bound catechol-O-methyltransferases.

Catechol-O-methyltransferase (COMT, EC 2.1.1.6) catalyzes the O-methylation of a wide array of catechol-containing substrates using s-adenosyl-L-methionine as the methyl donor. In the present study, we have cloned and expressed the human soluble and membrane-bound COMTs (S-COMT and MB-COMT, respectively) in Escherichia coli and have studied their biochemical characteristics for the O-methylation of representative classes of endogenous catechol substrates (catecholamines and catechol estrogens) as well as exogenous catechol substrates (bioflavonoids and tea catechins). Enzyme kinetic analyses showed that these two recombinant human COMTs are functionally active, with catalytic and kinetic properties nearly identical to those of crude or purified enzymes prepared from human tissues or cells. Kinetic parameters for the O-methylation of various substrates were characterized. In addition, computational modeling studies were conducted to better understand the molecular mechanisms for the different catalytic behaviors of human S- and MB-COMTs with respect to s-adenosyl-L-methionine, various substrates, and also the regioselectivity for the formation of mono-methyl ether products. Our modeling data showed that the binding energy values (Delta G) calculated for most substrates agreed well with the measured kinetic parameters. Also, our modeling data precisely predicted the regioselectivity for the O-methylation of these substrates at different hydroxyl groups, the predicted values matched nearly perfectly with the experimental data.