Characterization of the SARS-CoV-2 ExoN (nsp14ExoN-nsp10) complex: implications for its role in viral genome stability and inhibitor identification.

The SARS-CoV-2 coronavirus is the causal agent of the current global pandemic. SARS-CoV-2 belongs to an order, Nidovirales, with very large RNA genomes. It is proposed that the fidelity of coronavirus (CoV) genome replication is aided by an RNA nuclease complex, comprising the non-structural proteins 14 and 10 (nsp14-nsp10), an attractive target for antiviral inhibition. Our results validate reports that the SARS-CoV-2 nsp14-nsp10 complex has RNase activity. Detailed functional characterization reveals nsp14-nsp10 is a versatile nuclease capable of digesting a wide variety of RNA structures, including those with a blocked 3'-terminus. Consistent with a role in maintaining viral genome integrity during replication, we find that nsp14-nsp10 activity is enhanced by the viral RNA-dependent RNA polymerase complex (RdRp) consisting of nsp12-nsp7-nsp8 (nsp12-7-8) and demonstrate that this stimulation is mediated by nsp8. We propose that the role of nsp14-nsp10 in maintaining replication fidelity goes beyond classical proofreading by purging the nascent replicating RNA strand of a range of potentially replication-terminating aberrations. Using our developed assays, we identify drug and drug-like molecules that inhibit nsp14-nsp10, including the known SARS-CoV-2 major protease (Mpro) inhibitor ebselen and the HIV integrase inhibitor raltegravir, revealing the potential for multifunctional inhibitors in COVID-19 treatment.

Rare instances of haploid inducer DNA in potato dihaploids and ploidy-dependent genome instability.

In cultivated tetraploid potato (Solanum tuberosum), reduction to diploidy (dihaploidy) allows for hybridization to diploids and introgression breeding and may facilitate the production of inbreds. Pollination with haploid inducers (HIs) yields maternal dihaploids, as well as triploid and tetraploid hybrids. Dihaploids may result from parthenogenesis, entailing the development of embryos from unfertilized eggs, or genome elimination, entailing missegregation and the loss of paternal chromosomes. A sign of genome elimination is the occasional persistence of HI DNA in some dihaploids. We characterized the genomes of 919 putative dihaploids and 134 hybrids produced by pollinating tetraploid clones with three HIs: IVP35, IVP101, and PL-4. Whole-chromosome or segmental aneuploidy was observed in 76 dihaploids, with karyotypes ranging from 2n = 2x - 1 = 23 to 2n = 2x + 3 = 27. Of the additional chromosomes in 74 aneuploids, 66 were from the non-inducer parent and 8 from the inducer parent. Overall, we detected full or partial chromosomes from the HI parent in 0.87% of the dihaploids, irrespective of parental genotypes. Chromosomal breaks commonly affected the paternal genome in the dihaploid and tetraploid progeny, but not in the triploid progeny, correlating instability to sperm ploidy and to haploid induction. The residual HI DNA discovered in the progeny is consistent with genome elimination as the mechanism of haploid induction.

Active DNA Demethylation in Plants.

Methylation of cytosine (5-meC) is a critical epigenetic modification in many eukaryotes, and genomic DNA methylation landscapes are dynamically regulated by opposed methylation and demethylation processes. Plants are unique in possessing a mechanism for active DNA demethylation involving DNA glycosylases that excise 5-meC and initiate its replacement with unmodified C through a base excision repair (BER) pathway. Plant BER-mediated DNA demethylation is a complex process involving numerous proteins, as well as additional regulatory factors that avoid accumulation of potentially harmful intermediates and coordinate demethylation and methylation to maintain balanced yet flexible DNA methylation patterns. Active DNA demethylation counteracts excessive methylation at transposable elements (TEs), mainly in euchromatic regions, and one of its major functions is to avoid methylation spreading to nearby genes. It is also involved in transcriptional activation of TEs and TE-derived sequences in companion cells of male and female gametophytes, which reinforces transposon silencing in gametes and also contributes to gene imprinting in the endosperm. Plant 5-meC DNA glycosylases are additionally involved in many other physiological processes, including seed development and germination, fruit ripening, and plant responses to a variety of biotic and abiotic environmental stimuli.

Folate, genomic stability and colon cancer: The use of single cell gel electrophoresis in assessing the impact of folate in vitro, in vivo and in human biomonitoring.

Intake of folate (vitamin B9) is strongly inversely linked with human cancer risk, particularly colon cancer. In general, people with the highest dietary intake of folate or with high blood folate levels are at a reduced risk (approx. 25%) of developing colon cancer. Folate acts in normal cellular metabolism to maintain genomic stability through the provision of nucleotides for DNA replication and DNA repair and by regulating DNA methylation and gene expression. Folate deficiency can accelerate carcinogenesis by inducing misincorporation of uracil into DNA, by increasing DNA strand breakage, by inhibiting DNA base excision repair capacity and by inducing DNA hypomethylation and consequently aberrant gene and protein expression. Conversely, increasing folate intake may improve genomic stability. This review describes key applications of single cell gel electrophoresis (the comet assay) in assessing genomic instability (misincorporated uracil, DNA single strand breakage and DNA repair capacity) in response to folate status (deficient or supplemented) in human cells in vitro, in rodent models and in human case-control and intervention studies. It highlights an adaptation of the SCGE comet assay for measuring genome-wide and gene-specific DNA methylation in human cells and colon tissue.

DNA damage and genomic instability among workers formerly and currently exposed to asbestos.

Objectives Despite an asbestos ban in the European Union, exposure to asbestos still represents an occupational risk. Biomarkers of DNA damage and genomic instability in groups exposed to asbestos may contribute to the identification of subgroups/subjects at higher risk. Methods A cross-sectional study was conducted on 468 male individuals (80 working in occupational settings with potential exposure to asbestos fibers, 202 retired workers with past exposure, and 186 non-exposed controls) to compare genomic instability, cell proliferation and differentiation level using the non-invasive micronucleus buccal cytome assay. Data on demographic variables, lifestyle, and occupational history were collected with a standardized questionnaire. Micronuclei (MN) and other biomarkers of DNA damage and genomic instability were scored in a minimum of 2000/1000 cells per individual, respectively. Results Univariate and multivariate analysis showed opposite associations of MN frequency with current and former exposure. Compared to unexposed controls, workers with current potential exposure to asbestos had 55% lower MN frequency [95% confidence interval (CI) 71-29%, P<0.001] while those with past exposure had 34% higher MN frequency (95% CI 1-77%, P<0.001). The frequency of cells with condensed chromatin and binucleated cells was elevated among formerly exposed workers. The multivariate analysis did not reveal any actual confounders, although lower MN frequency was observed among subjects eating fresh fruit or vegetables every day or taking vitamin supplements. Conclusions Active workers with potential exposure to asbestos fibers did not show increased genomic damage. On the contrary, workers exposed in the past experienced a persistently elevated genomic instability, which may be used for risk assessment at subgroup or individual level.

Annealing of Complementary DNA Sequences During Double-Strand Break Repair in Drosophila Is Mediated by the Ortholog of SMARCAL1.

DNA double-strand breaks (DSBs) pose a serious threat to genomic integrity. If unrepaired, they can lead to chromosome fragmentation and cell death. If repaired incorrectly, they can cause mutations and chromosome rearrangements. DSBs are repaired using end-joining or homology-directed repair strategies, with the predominant form of homology-directed repair being synthesis-dependent strand annealing (SDSA). SDSA is the first defense against genomic rearrangements and information loss during DSB repair, making it a vital component of cell health and an attractive target for chemotherapeutic development. SDSA has also been proposed to be the primary mechanism for integration of large insertions during genome editing with CRISPR/Cas9. Despite the central role for SDSA in genome stability, little is known about the defining step: annealing. We hypothesized that annealing during SDSA is performed by the annealing helicase SMARCAL1, which can anneal RPA-coated single DNA strands during replication-associated DNA damage repair. We used unique genetic tools in Drosophila melanogaster to test whether the fly ortholog of SMARCAL1, Marcal1, mediates annealing during SDSA. Repair that requires annealing is significantly reduced in Marcal1 null mutants in both synthesis-dependent and synthesis-independent (single-strand annealing) assays. Elimination of the ATP-binding activity of Marcal1 also reduced annealing-dependent repair, suggesting that the annealing activity requires translocation along DNA. Unlike the null mutant, however, the ATP-binding defect mutant showed reduced end joining, shedding light on the interaction between SDSA and end-joining pathways.

Yin Yang 1 (YY1): Regulation of Survivin and Its Role In Invasion and Metastasis.

Despite significant clinical and basic science advancements, cancer remains a devastating disease that affects people of all ages, races, and backgrounds. The pathogenesis of cancer has recently been described to result from eight biological capabilities or hallmarks and two enabling characteristics. These eight hallmarks are: deregulation of cellular energetics, avoiding immune destruction, enabling replicative immortality, inducing angiogenesis, sustaining proliferative signaling, evading growth suppressors, resisting cell death, and activating invasion and metastasis. The enabling characteristics are: genome instability and mutation and tumor-promoting inflammation. Survivin, the fourth most common transcript found in cancer cells, is a protein that is thought to be involved in the enhanced proliferation, survival, and metastasis and possibly other key hallmarks of cancer cells. Understanding how this gene is turned on and off is vitally important for attempt improving cancer management and therapy. Our work has identified a novel transcriptional regulator of survivin called Yin Yang 1 (YY1), which has been observed to activate some gene promoters and repress others and is gaining increasing interest as a target of cancer therapy. Our work shows for the first time that YY1 represses survivin transcription by physically interacting with the survivin promoter. Furthermore, YY1 appears to contribute to basal survivin transcriptional activity, indicating that disruption of its binding may in part contribute to survivin overexpression after cellular stress events including chemotherapy and radiotherapy.

Genomic instability in human cancer: Molecular insights and opportunities for therapeutic attack and prevention through diet and nutrition.

Genomic instability can initiate cancer, augment progression, and influence the overall prognosis of the affected patient. Genomic instability arises from many different pathways, such as telomere damage, centrosome amplification, epigenetic modifications, and DNA damage from endogenous and exogenous sources, and can be perpetuating, or limiting, through the induction of mutations or aneuploidy, both enabling and catastrophic. Many cancer treatments induce DNA damage to impair cell division on a global scale but it is accepted that personalized treatments, those that are tailored to the particular patient and type of cancer, must also be developed. In this review, we detail the mechanisms from which genomic instability arises and can lead to cancer, as well as treatments and measures that prevent genomic instability or take advantage of the cellular defects caused by genomic instability. In particular, we identify and discuss five priority targets against genomic instability: (1) prevention of DNA damage; (2) enhancement of DNA repair; (3) targeting deficient DNA repair; (4) impairing centrosome clustering; and, (5) inhibition of telomerase activity. Moreover, we highlight vitamin D and B, selenium, carotenoids, PARP inhibitors, resveratrol, and isothiocyanates as priority approaches against genomic instability. The prioritized target sites and approaches were cross validated to identify potential synergistic effects on a number of important areas of cancer biology.

Inducing pluripotency in vitro: recent advances and highlights in induced pluripotent stem cells generation and pluripotency reprogramming.

Induced pluripotent stem cells (iPSCs) are considered patient-specific counterparts of embryonic stem cells as they originate from somatic cells after forced expression of pluripotency reprogramming factors Oct4, Sox2, Klf4 and c-Myc. iPSCs offer unprecedented opportunity for personalized cell therapies in regenerative medicine. In recent years, iPSC technology has undergone substantial improvement to overcome slow and inefficient reprogramming protocols, and to ensure clinical-grade iPSCs and their functional derivatives. Recent developments in iPSC technology include better reprogramming methods employing novel delivery systems such as non-integrating viral and non-viral vectors, and characterization of alternative reprogramming factors. Concurrently, small chemical molecules (inhibitors of specific signalling or epigenetic regulators) have become crucial to iPSC reprogramming; they have the ability to replace putative reprogramming factors and boost reprogramming processes. Moreover, common dietary supplements, such as vitamin C and antioxidants, when introduced into reprogramming media, have been found to improve genomic and epigenomic profiles of iPSCs. In this article, we review the most recent advances in the iPSC field and potent application of iPSCs, in terms of cell therapy and tissue engineering.

Genomic imprinting, action, and interaction of maternal and fetal genomes.

Mammalian viviparity (intrauterine development of the fetus) introduced a new dimension to brain development, with the fetal hypothalamus and fetal placenta developing at a time when the fetal placenta engages hypothalamic structures of the maternal generation. Such transgenerational interactions provide a basis for ensuring optimal maternalism in the next generation. This success has depended on genomic imprinting and a biased role of the matriline. Maternal methylation imprints determine parent of origin expression of genes fundamental to both placental and hypothalamic development. The matriline takes a further leading role for transgenerational reprogramming of these imprints. Developmental errors are minimized by the tight control that imprinted genes have on regulation of downstream evolutionary expanded gene families important for placental and hypothalamic development. Imprinted genes themselves have undergone purifying selection, providing a framework of stability for in utero development with most growth variance occurring postnatally. Mothers, not fathers, take the lead in the endocrinological and behavior adaptations that nurture, feed, and protect the infant. In utero coadaptive development of the placenta and hypothalamus has thus required a concomitant development to ensure male masculinization. Only placental male mammals evolved the sex determining SRY, which activates Sox9 for testes formation. SRY is a hybrid gene of Dgcr8 expressed in the developing placenta and Sox3 expressed in hypothalamic development. This hybridization of genes that take their origin from the placenta and hypothalamus has enabled critical in utero timing for the development of fetal Leydig cells, and hence testosterone production for hypothalamic masculinization.

Use of pantothenate as a metabolic switch increases the genetic stability of farnesene producing Saccharomyces cerevisiae.

We observed that removing pantothenate (vitamin B5), a precursor to co-enzyme A, from the growth medium of Saccharomyces cerevisiae engineered to produce ß-farnesene reduced the strain׳s farnesene flux by 70%, but increased its viability, growth rate and biomass yield. Conversely, the growth rate and biomass yield of wild-type yeast were reduced. Cultivation in media lacking pantothenate eliminates the growth advantage of low-producing mutants, leading to improved production upon scale-up to lab-scale bioreactor testing. An omics investigation revealed that when exogenous pantothenate levels are limited, acyl-CoA metabolites decrease, ß-oxidation decreases from unexpectedly high levels in the farnesene producer, and sterol and fatty acid synthesis likely limits the growth rate of the wild-type strain. Thus pantothenate supplementation can be utilized as a "metabolic switch" for tuning the synthesis rates of molecules relying on CoA intermediates and aid the economic scale-up of strains producing acyl-CoA derived molecules to manufacturing facilities.

Evaluation of mutagenic and genotoxic activities of lobeline and its modulation on genomic instability induced by ethanol.

AIM: Lobeline is a natural alkaloid derived from Lobelia inflata that has been investigated as a clinical candidate for the treatment of alcoholism. In a pre-clinical trial, lobeline decreased the preference for and consumption of ethanol, due to the modulation of the nicotinic acetylcholine receptor. However, the interaction between lobeline and ethanol is poorly known and thus there are safety concerns. The present study was conducted to evaluate the mutagenic and genotoxic effects of lobeline and assess its modulation of ethanol-induced toxicological effects. MAIN METHODS: CF-1 male mice were divided into five groups. Groups received an intraperitoneal injection of saline solution, lobeline (5 or 10mg/kg), ethanol (2.5 g/kg), or lobeline plus ethanol, once a day for three consecutive days. Genotoxicity was evaluated in peripheral blood using the alkaline comet assay. The mutagenicity was evaluated using both Salmonella/microsome assay in TA1535, TA97a, TA98, TA100, and TA102 Salmonella typhimurium strains and the micronucleus test in bone marrow. Possible liver and kidney injuries were evaluated using biochemical analysis. KEY FINDINGS: Lobeline did not show genotoxic or mutagenic effects and did not increase the ethanol-induced genotoxic effects in blood. Lobeline also protected blood cells against oxidative damage induced by hydrogen peroxide. Biochemical parameters were not altered, indicating no liver or kidney injuries or alterations in lipid and carbohydrate metabolisms. SIGNIFICANCE: These findings suggest that lobeline does not induce gene or chromosomal mutations, and that this lack of genetic toxicity is maintained in the presence of ethanol, providing further evidence of the safety of this drug to treat alcohol dependence.

Folate deficiency is not associated with increased mitochondrial genomic instability: results from dietary intake and lymphocytic mtDNA 4977-bp deletion in healthy young women in Italy.

The mitochondrial DNA (mtDNA) 4977-bp deletion is a biomarker of mitochondrial genomic instability. It is frequently detected in a number of sporadic diseases, and it accumulates in many tissues during aging. Folic acid plays an important role in the maintenance of genomic stability in mammals. The aim of the present cross-sectional study was to characterise the levels of the mtDNA deletion in the lymphocytes of healthy young women, taking into account folate intake, red blood cell (RBC) folate levels and the distribution of the methylenetetrahydrofolate reductase (MTHFR) gene C677T polymorphism. Folate intake was estimated by a food frequency questionnaire. Determination of the MTHFR C677T polymorphism and of the mtDNA deletion was performed by real-time polymerase chain reaction analysis. A total of 476 women were enrolled. Low levels of deletion were found (mean ΔCt = 1.24). After multivariate analysis, results did not show any significant relationship between age, smoking habits, pregnancy status, nutritional status, inadequate folate intake, folate deficiency, use of folic acid supplements, MTHFR C677T polymorphism and mtDNA 4977-bp deletions. The lack of association between inadequate folate intake, folate deficiency and mitochondrial genomic instability was confirmed also considering reference values of folate based on DNA damage prevention. Our results indicate that mtDNA 4977-bp deletions are maintained at low levels in lymphocytes of young healthy women despite the wide range of variation of folate intakes and folate status. Future studies, carefully designed to address limits and methodological issues related to variation of this biomarker as an effect of different dietary patterns and of folate status, could provide further insight on the specific mechanisms that are acting in lymphocytes of healthy subjects under observed folate intake.

Evidence for a role of Arabidopsis CDT1 proteins in gametophyte development and maintenance of genome integrity.

Meristems retain the ability to divide throughout the life cycle of plants, which can last for over 1000 years in some species. Furthermore, the germline is not laid down early during embryogenesis but originates from the meristematic cells relatively late during development. Thus, accurate cell cycle regulation is of utmost importance to avoid the accumulation of mutations during vegetative growth and reproduction. The Arabidopsis thaliana genome encodes two homologs of the replication licensing factor CDC10 Target1 (CDT1), and overexpression of CDT1a stimulates DNA replication. Here, we have investigated the respective functions of Arabidopsis CDT1a and CDT1b. We show that CDT1 proteins have partially redundant functions during gametophyte development and are required for the maintenance of genome integrity. Furthermore, CDT1-RNAi plants show endogenous DNA stress, are more tolerant than the wild type to DNA-damaging agents, and show constitutive induction of genes involved in DNA repair. This DNA stress response may be a direct consequence of reduced CDT1 accumulation on DNA repair or may relate to the ability of CDT1 proteins to form complexes with DNA polymerase ε, which functions in DNA replication and in DNA stress checkpoint activation. Taken together, our results provide evidence for a crucial role of Arabidopsis CDT1 proteins in genome stability.

Supplemental dietary folic acid has no effect on chromosome damage in erythrocyte progenitor cells of mice.

Folate deficiency can cause chromosome damage, which could result from reduced de novo thymidylate synthesis or DNA hypomethylation. High folic acid intake has been hypothesized to inhibit folate-dependent one-carbon metabolism, which could also lead to DNA damage. A large proportion of the general population may have high folic acid intakes. In this study, 2 experiments were conducted to examine the effects of folate on chromosome damage. First, male mice were fed folic acid-deficient (D) (0 mg folic acid/kg diet), control (C) (2 mg/kg), or folic acid-supplemented (S) (6 mg folic acid/kg diet) diets from weaning to maturity. Second, female mice were fed the D, C, or S diet throughout pregnancy, lactation, and breeding for 3 generations; male mice from the F3 generation were fed the same diet as their mothers from weaning, producing D, C, and S F3 male mice. RBC micronucleus frequencies, a measure of chromosome damage or aneuploidy, were determined for both experimental groups. In mice fed diets from weaning to maturity, erythrocyte micronucleus frequency was 24% greater in D compared with C mice. F3 mice fed diet D had 260% and 174% greater reticulocyte and erythrocyte micronucleus frequencies compared with F3 C mice, respectively. The S diets did not affect micronucleus frequency, suggesting that excess folic acid at this level does not promote or protect against chromosome damage. The results suggest that chronic exposure to folic acid at the levels similar to those achieved through fortification is unlikely to be clastogenic or aneugenic.

Increased frequency of micronuclei in lymphocytes of infertile males after exposure to gamma irradiation: a possible sign of genomic instability.

PURPOSE: Induced chromosomal instability and micronucleus (MN) formation in blood lymphocytes of infertile men in comparison with fertile men exposed to gamma radiation was investigated. METHODS: Blood samples of healthy and infertile donors were irradiated by 2 and 4 Gy Co-60 gamma-rays, then cultured in RPMI-1640 complete medium containing 1% phytoheamaglutinin (PHA) and incubated in a CO(2) incubator. Cytochalasin-B was added to the cultures at a final concentration of 4 µg/ml. Finally, harvesting, slide making, and analysis were performed according to standard procedures. RESULTS: We observed a statistically significant difference between the frequencies of micronuclei in lymphocytes of infertile individuals, compared to healthy donors, before and after exposure to gamma rays. Although higher in azoospermia patients, the frequency of MN was not statistically different between infertile groups. CONCLUSIONS: This study indicates that genomic instability in infertile men could probably contribute to the development of an impaired reproductive capacity.

[Study on preservation of Fritillaria anhuiensis by vitrification in vitro].

OBJECTIVE: To establish cryopreservation system of shoot-tips from Fritillaria anhuiensis. METHOD: Taking vitrification as system of cryopreservation, the shoot tips with length 2-3 mm were precultured in MS medium enriched with 0.4 mol x L(-1) sucrose for 3 d. They were treated for 20 min with 60% PVS2 at 25 degrees C, and then subjected to ice-cooled vitrification solution for 60 min and transferred to 2 mL cryotubes with fresh vitrification solution (PVS2) and plunged into liquid nitrogen. After rapid thawing in 40 degrees C water bath for 1 min, shoot-tips were expelled into MS medium containing 1.2 mol x L(-1) sucrose for 20 min. Further recovery and growth took place on regeneration medium in the dark at 25 degrees C for 2 weeks, and then with light/dark cycle of 12/12 h. The genetic integrity of cryopreserved shoot-tips was identified through products of PCR with arbitrary primers. RESULT AND CONCLUSION: The highest survival rates of shoot-tips reached 79.9% by vitrification, and the regeneration rates were 52.3%. No changes were found between treated materials and untreated materials in genomic DNA.

Preconceptional paternal exposure to depleted uranium: transmission of genetic damage to offspring.

Depleted uranium (DU) is an alpha particle emitter and radioactive heavy metal used in military applications. Due to internalization of DU during military operations and the ensuing chronic internal exposure to DU, there are concerns regarding its potential health effects. Preconceptional paternal irradiation has been implicated as a causal factor in childhood cancer and it has been suggested that this paternal exposure to radiation may play a role in the occurrence of leukemia and other cancers to offspring. Similarly, in vivo heavy metal studies have demonstrated that carcinogenic effects can occur in unexposed offspring. Using a transgenic mouse system employing a lambda shuttle vector allowing mutations (in the lacI gene) to be analyzed in vitro, we have investigated the possibility that chronic preconceptional paternal DU exposure can lead to transgenerational transmission of genomic instability. The mutation frequencies in vector recovered from the bone marrow cells of the F1 offspring of male parents exposed to low, medium, and high doses of internalized DU for 7 mo were evaluated and compared to control, tantalum, nickel, and gamma radiation F1 samples. Results demonstrate that as paternal DU-dose increased there was a trend towards higher mutation frequency in vector recovered from the DNA obtained from bone marrow of F1 progeny; medium and high dose DU exposure to P1 fathers resulted in a significant increase in mutation frequency in F1 offspring (3.57 +or - 0.37 and 4.81 + or - 0.43 x 10; p < 0.001) in comparison to control (2.28 + or - 0.31 x 10). The mutation frequencies from F1 offspring of low dose DU, Ta- or Ni-implanted fathers (2. 71 + or - 0.35, 2.38 + or - 0.35, and 2.93 + or - 0.39 x 10, respectively) were not significantly different than control levels (2.28 + or - 0.31 x 10). Offspring from Co (4 Gy) irradiated fathers did demonstrate an increased lacI mutation frequency (4.69 + or - 0.48 x 10) as had been shown previously. To evaluate the role of radiation involved in the observed DU effects, males were exposed to equal concentrations (50 mg U L) of either enriched uranium or DU in their drinking water for 2 mo prior to breeding. A comparison of these offspring indicated that there was a specific-activity dependent increase in offspring bone marrow mutation frequency. Taken together these uranyl nitrate data support earlier results in other model systems showing that radiation can play a role in DU-induced biological effects in vitro. However, since the lacI mutation model measures point mutations and cannot measure large deletions that are characteristic of radiation damage, the role of DU chemical effects in the observed offspring mutation frequency increase may also be significant. Regardless of the question of DU-radiation vs. DU-chemical effects, the data indicate that there exists a route for transgenerational transmission of factor(s) leading to genomic instability in F1 progeny from DU-exposed fathers.

Cryopreservation of in vitro grown shoot tips and apical meristems of the forage legume Arachis pintoi.

A cryopreservation protocol using the encapsulation-dehydration procedure was established for shoot tips (2-3 mm in length) and meristems (0.3-0.5 mm) sampled from in vitro plantlets of diploid and triploid cytotypes of Arachis pintoi. The optimal protocol was the following: after dissection, explants were precultured for 24 h on establishment medium (EM), encapsulated in calcium alginate beads and pretreated in liquid EM medium with daily increasing sucrose concentration (0.5, 0.75, 1.0 M) and desiccated to 22-23 percent moisture content (fresh weight basis). Explants were frozen using slow cooling (1 C per min from 25C to -30C followed by direct immersion in liquid nitrogen), thawed rapidly and post-cultured in liquid EM medium enriched with daily decreasing sucrose concentrations (0.75, 0.50, 0.1 M). Explants were then transferred to solid EM medium in order to achieve shoot regeneration, then on Murashige and Skoog medium supplemented with 0.05 microM naphthalene acetic acid to induce rooting of shoots. With this procedure, 53 percent and 56 percent of cryopreserved shoot tips of the diploid and triploid cytotypes, respectively, survived and formed plants. However, only 16 percent of cryopreserved meristems of both cytotypes regenerated plants. Using ten isozyme systems and seven RAPD profiles, no modification induced by cryopreservation could be detected in plantlets regenerated from cryopreserved material.

What is the role and impact of molecular markers on treatment decisions for colorectal cancer in the adjuvant setting?

The new mantra for delivering optimal cancer treatment is "personalized care." This extends beyond the holistic to using germline and somatic tumoral mutations to link a specific therapy to some prognostic or predictive factor which defines a particularly responsive patient subgroup who might benefit most from treatment. Furthermore, inherited polymorphisms have the potential to greatly modulate the side effects of treatment, especially for chemotherapy which has a notoriously narrow therapeutic window.