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The final reaction products were separated by urea holb? PAGE Hlb?k were exposed to the phosphor-imager screen. A Fujifilm phosphor-image scanner was used to visualize the bands. Counts of the bands, or the desired stretches of the lanes, were recorded for further analyses. A minimum of three experiments were performed for each reaction to get the average counts and standard deviations. The relative ligation frequency RLFi. Nick translation, single nucleotide and 10 nucleotides cleavage rates were calculated from the initial slopes of the fitted curves, as described in the S1 Text.
Summary: Origin recognition complex subunit 2
The BrdU cross-linking assay The assay was designed with modifications to the protocol mentioned [ 46 ]. Since the nick holb?kk reaction was observed to reach completion within a minute Uolb?k 1we allowed crosslinking for one minute. Holbk reaction mixture was loaded on a Edsting gel to ho,b?k the band shift. Structural modeling of E. Supporting information S1 Fig. Memorex dvd player hook up shift assay shows correct primer annealing during preparation of template. The native gel shows the separation of 19 nucleotides Edatihg 128 nucleotides and 67 nucleotides lane 3 long oligonucleotides.
However, Erating oligonucleotides 19 and 28 bases did not found to be annealed lane Eating. On the other hand, the 67 bases long oligonucleotide, which has complementary regions for both 19 and 28 bases long oligonucleotides, has also completely holb? to these 19 and 28 bases oligonucleotides lanes 6 and 7, respectively. The fraction of the Edafing counts that appeared at 67 nucleotide position were plotted. The rate of the uolb?k translation through RNA substrate was calculated Edating holb?k the fitted curve shown above also see S1 Text.
A The lanes 2—5 are showing purified individual proteins that are used in the pull down assay. Food Holb?i and Risk Assessment Food toxicology defines the science dealing nolb?k the toxicological effects of food constituents [ 1 ]. Indeed, food and feed are Eating the most complex cocktails of low-molecular weight xenobiotics to which hllb?k living organisms are exposed [ 2 ]. Extensive Edafing on the nature and the route of food and feed contamination would be out of the scope Edaying detailed information can be found in [ 4 ]. However, it is worthy to quote that the naturally occurring contaminants, i.
Among them, mycotoxins have been object of a growing number of investigations in the last years as they may severely threaten human and Eadting health [ 6 ]. The overall burden of mycotoxins encompasses medical costs, financial detriments and food waste [ 7Edatinb ]. Therefore, many countries have adopted regulations hlb?k limit the overall mycotoxin dietary exposure, safeguarding the Edatiny and animal health. At the same time, the setting of regulatory actions must pursue tricky tradeoffs to avoid unreasonable food wasting hlob?k to limit trade frictions [ 9 ]. In order hollb?k address such a premise, the regulatory actions have to Edatinb on sound risk assessment studies that must be based on a solid background of toxicological knowledge.
So far, risk assessments studies have been carried out on some major mycotoxins from the public health perspective i. Obviously, the deeper the toxicological understanding, the more appropriate, precise and effective the risk assessment of a molecule or a class of molecules can be. In this regard, however, many aspects in the toxicology of mycotoxins are not fully understood yet, especially from a mechanistic point of view. This ultimately poses a degree of uncertainty to the current risk statements [ 10 ]. In addition, beside the few mycotoxins regulated so far, many more can contaminate food and feed.
Alternariol and alternariol methyl ether, enniatins, moniliformin, beauvaricin, culmorin and fusaric acid are among those gathering wide attention [ 11 ]. However, the shortage of toxicological data and the lack of a convincing consensus on toxicity and on the mechanisms of action prevent from setting appropriate guidelines. Overall, this scenario points out the need to move further steps in understanding mycotoxins toxicology to timely fill the data gaps and, therefore, to provide a sufficiently informed scenario for risk assessment. Keeping in mind that the full comprehension of mycotoxins toxicology is a major challenge for the Scientific Community and it requires inter-disciplinary approaches, this work focuses on the possible support offered by computational methods in the field of toxicodynamics, moving from principles and potentialities Section 2 and Section 3 to the application to case-studies Section 4 and outlooks Section 5.
Toxicokinetics and Toxicodynamics in Chemicals Risk Assessment Risk assessment is referred to as the stepwise and iterative process aimed at estimating from qualitative or quantitative point of view the likelihood, or the magnitude and probability, of harmful effects to individuals or population due to a given chemical or group of chemicals. Briefly, the main steps in chemical risk assessment are the following [ 12 ]: Typically, in the chemicals risk assessment, the hazard identification and hazard characterization rely on extrapolations from toxicological data resulting from in vitro or, better, in vivo animal studies. However, understanding the relationship between the mechanistic aspects of toxic action and the time-dependency of toxicity is an undeniable prerequisite to undergo meaningful risk assessment studies [ 13 ].
This relationship is typically explained from a toxicokinetics and toxicodynamics perspective. Toxicokinetics Toxicokinetics determines the relationship between the concentration of a given chemical to which an individual is exposed, and the concentration of toxicologically active compounds at the district of action, intended as the organ, tissue, cellular or molecular locus where the chemicals exert the effect. While multiple routes of exposure may exist for mycotoxins e. Therefore, in this case, the differences between the ingested concentration and the concentration at the site of action are explained by the LADME paradigm [ 16 ].
While dietary habits are crucial for the definition of the exposure to food contaminants, the persistence of toxicologically active compounds in the living organisms depends on the ADME kinetics and eventual bioaccumulation phenomena [ 6 ]. Toxicodynamics Toxicodynamics determines the relationship between the concentration of a toxicant at the site of action and the toxic effect at the level of molecule, cell, tissue, organ or organism. Typically, the receptors are proteins, but they can be also other macromolecules such as DNA- or RNA-protein complexes and glycoproteins. The receptor binding is the primal molecular event of non-genotoxic compounds action determining the so defined molecular initiating event MIE [ 20 ].
Each causally connected event from the receptor binding to the final adverse outcome is linearly connected defining the adverse outcome pathway AOP [ 23 ]. Concerning the early step of MIE, non-genotoxic compounds establish a dynamic binding with the receptors reaching the steady-state equilibrium in a given time frame, depending on the kinetic of association and dissociation [ 24 ]. From a molecular point of view, this binding event typically determines structural changes altering the normal receptor structure and functionality, and leading thus to perturbation of homeostasis.
The toxic outcome onsets when such perturbation exceeds a threshold that cannot be tolerated. The relative concentration of the bound receptor at the site of action strongly influences the intensity in triggering the toxic stimulus. In turn, the relative concentration of bound receptor depends on the concentration of toxicant at the site of action, its binding affinity and the kinetics of association and dissociation. On this basis, it can be argued that the strength of interaction namely, the thermodynamic favor of the ligand-receptor complex formation can proportionally correlate to the potency in triggering the early step of toxic stimulus. Therefore, in the context of the chemical risk assessment process, toxicodynamics investigations focused on the receptor binding may support both steps of hazard identification and characterization by probing the primal molecular event of toxic action vide infra.
For many years, the toxicological understanding of mycotoxins including the dynamics aspects has been based mainly on the independent assessment of few reference molecules representing broad classes of compounds i.
Nowadays, however, food myco-toxicologists and risk assessors are moving toward a group-based approach to better address Edating holb?k multi-toxins exposure paradigm e. In this framework, the risk related to mycotoxins exposure should be assessed considering the combined action of multiple compounds considering either the simultaneous exposure to different chemo-types i. Indeed, food and Edating holb?k are typically a source of multiple toxins as they may contain not only different mycotoxins chemo-types, but also many metabolites for each class of toxin. Even though most of these metabolites and analogues are still lacking of a sufficient toxicological characterization, some of them have proved to be stronger than the parent compounds in triggering the molecular cascade underlying the AOP.
Therefore, it can be argued that the relative abundance of the differently active metabolites at the site of action may have a relevant role in determining the magnitude of toxic stimulus triggering, with possible consequences also on the magnitude of the overall toxic effect. On this basis, the toxicodynamics of mycotoxins may strongly depend on the overall concentration at the site of action of all the mycotoxins metabolites and analogues that are able to bind the receptor. Contextually, toxic equivalents are referred to as those molecules that may occur at the site of action and may act as functional analogues in binding a specific receptor.
Accordingly, the understanding of the dynamic aspects of mycotoxins toxicity ideally requires a comprehensive assessment of all the toxic equivalents that can be found at the site of action. Following this principle, EFSA has recently provided health-based guidelines for zearalenone considering the relative potency factors of the main modified forms [ 26 ]. Extending such approach to the other mycotoxins poses a major challenge. Indeed, the relative contribution of each toxic analogue cannot be easily extrapolated from the in vitro and in vivo toxicological data.