To begin hypothesis-testing, we perform a focused DUE for an unremediated, Pasadena, California toxic site. Our DUE methods are (a) comparing project-specific, remediation-assessment data with the remediation-assessment conceptual site model (CSM), in order to identify data gaps, and (b) using data-gap directionality to assess possible determinate bias . Our results reveal (1) major CSM data gaps, particularly regarding Pasadena-toxic-site risks to children; (2) determinate bias, namely, risk underestimation; thus (3) likely inadequate remediation. Our discussion shows that if these results are generalizable, requiring routine, independent, DUEs might deter flawed toxic-site assessment/cleanup and resulting health threats, especially to children.Nearly 25 percent of US children live within 2 km of toxic-waste sites, most of which are in urban areas. They face higher rates of cancer than adults, partly because the dominant contaminants at most US hazardous-waste sites include genotoxic carcinogens, like trichloroethylene, that are much more harmful to children. The  By the year 2035, the US Environmental Protection Agency (EPA) projects  that 350,000 of the nation’s toxic-waste sites will need remediation, including military, other federal, state, and private sites . Most arAcross the US, tens of millions of people also are at risk because of contaminated drinking water. In more than half the nation’s public-drinking-water systems, levels of the genotoxic carcinogen trichloroethylene (TCE) exceed what is safe for children’s health ,9.Why are children more vulnerable to such contaminants? Children and pregnant women are more vulnerable than adults to toxic wastes because fetuses and children have special windows of increased vulnerability during different developmental periods. Exposures during these critical windows could cause permanent structural, organ-system, or functional deficits, such as TCE-induced heart defects from maternal, first-trimester, pregnancy exposure. Fetuses and children also experience rapid growth and development, thus a higher rate of cell division and differentiation, and have less developed immunological and detoxification systems, and more efficient mechanisms for blood-brain transport of contaminants—all of which may increase their susceptibility. Children likewise receive greater toxic exposures, per kilogram of body weight than adults, because they take in proportionately more food, water, and air. In addition, younger children have greater (than adult) pollutant exposures because of lactation, hand-to-mouth activity, and a breathing zone closer to soil contaminants. Because children have a longer lifetime to express pollutant damage, they also experience greater harm than adults from contaminants  (p (pfirst victims)   ; [74] (pvictims)   .second DUE sub-method, determining directionality of any data gaps, so as to assess any determinate bias, includes 3 main steps. These are (1) identifying each data gap, if any; (2) using site data to determine whether reported risk values are lower/higher than they likely are and whether assessors ignored any above-allowed-level risks; and (3) assessing each data gap for its contribution to risk under/over-estimates, thus to directionality and determinate bias  areas and …depths”  , inhalation, through (7) air, via (3) contaminant volatilization to soil vapor. This gap arises—as the health-risk calculation includes only indoor risks from VOC vapor intrusion—no other risks, as from ambient-air, soil-gas exposure. Yet carcinogenic-gas risks are extraordinarily high, up to nearly 750,000 times above allowed levels  see the.The assessors’ justification for ignoring soil-gas exposures—and leaving data gaps—is their claim that “VOCs in outdoor (ambient) air emanating from soil are not expected to pose a health risk”  (p. 31).First, the ambient-air-VOCs data gap in site assessments ignores the child-health alerts of the US ATSDR. ATSDR has repeatedly warned that ambient air is one of the two “most important routes of exposure” to TCE “for most members of the general population,” especially for “people living in the vicinity of a hazardous waste site” ,  could remain in place at concentrations that may exceed some regulatory screening levels”; carcinogens will remain because the only required pre-construction removal is of 11 small metals “hotspots”  vapor intrusion until such time that soil vapor is removed which could be more than a year”  cannot take groundwater into account at this time” [is time”  (p. 35).is time”  (p. 6). Second, site assessors attempted to explain their not performing groundwater-risk assessment by saying “We have not been able to obtain recent analytical data from the three wells” near the site [the site  (p. 11).the site . The senThird, assessors attempted to explain their failure (to perform groundwater testing) by claiming it was unnecessary, given that depth to groundwater is greater than 91.4 m, and groundwater is not “a planned source of drinking water” [g water”  (p. 31).g water”  (p. 7). g water”   soil contamination, nearly a million times higher than allowed ,  testing  (p. 12). testing . This re testing  (p. 9).In addition, contaminants cannot be removed, post-construction, as the assessors claim, because even limited site testing and isoconcentration maps (for only 2 of the scores of contaminants) cover at least one-fourth of the site ,67, and Fifth, assessors tried to explain not performing preconstruction, groundwater testing/assessment by claiming that because it would interfere with site construction/earthmoving, it must be done later [ne later  (p. 32).scientific justification for failure to assess groundwater.The developer/assessors admitted their questionable priorities in site documents. They said later groundwater-testing wells must be placed, not in areas where scientifically rigorous sampling requires placement, but where they “will not impact planned site development”  (p. 7). Sixth, the site developer tried to explain not performing preconstruction-site-groundwater testing by claiming that the Prospective Purchase Agreement (PPA), that Trammell Crow obtained from California DTSC [nia DTSC , did notnia DTSC  (p. 10).nia DTSC . This TrHowever, the PPA does not change the fact that state and federal cleanup rules require groundwater testing/assessment (see preceding paragraphs). Moreover, the PPA itself requires that “intended uses of the site  will not pose health risks to persons,” and that the developer will “reduce the human health risk to acceptable levels.” Yet if previous arguments are correct, closing the groundwater-testing gap is necessary to meet both PPA requirements   to ignore state/federal toxic-cleanup standards. However, as At least two possible explanations come to mind for the second, or airborne-risk, data gap. One reason may be that because no one did a DUE for site-remediation documents, the airborne/soil-borne data gaps were not obvious. The documents did claim that sampling data collected in 2006 were “usable for their intended purposes” because they met data-quality criteria such as adequate sample preservation and detection limits   do . DTSC alPerhaps another reason Pasadena-remediation assessments have air/soil-borne data gaps is that such gaps are common in toxic-site cleanups, especially wherever DUEs are not required. For instance, Trammell Crow is the largest US commercial real-estate developer, with assets of more than $65 billion . Its “BrAnother possible explanation for Trammell Crow’s data gaps is financial conflicts of interest. Since at least the 1980s social-science research has clearly established “the funding effect,” that research conclusions are likely to serve the interests of those who fund the research, an effect that is statistically correlated with research outcomes. Thus, if private companies fund research on safety of their chemicals, drugs, or site remediation, the research is likely to show safety. If nonprofit, university, or government agencies fund the same research, it is much less likely to show safety. This “funding effect” has been overwhelmingly established in environmental-health, tobacco, climate-change, chemical-toxicity studies, and so on; this effect also could be occurring in Pasadena toxics-remediation assessments by those whose profits are at stake ,174,175.Pasadena city officials were the first to approve site-remediation documents, but at least three factors could have encouraged their approving almost any housing development—even on an inadequately assessed toxic site. These three factors are California’s housing shortage , PasadenAnother factor in the approval of scientifically deficient toxic-site-remediation assessments is that the Pasadena mayor (and developer) Terence Tornek and many city-council members accept campaign donations from developers—whose local projects they vote to approve/disapprove . However10-times-higher cancer risks (than adults will) from the toxic site, yet assessors performed no child risk assessment to quantify/assesses any of these higher risks  are most at risk from data gaps in Pasadena toxic-site-assessment/remediation documents. These younger children:Given these heightened toxic-site threats to children, assessors may have failed to protect children because they tend to be natural scientists/engineers, thus unaware of child-health threats. Public officials also often trust engineering experts, not realizing they have little pediatric expertise.In particular, assessors/officials may not know that specific social problems put children at greater risk from toxic sites. These already-noted problems include the facts that disproportionate numbers of vulnerable poor people, minorities, and children live near toxic-waste sites ,180,181;For instance, children conceived by mothers living within 3 km of an unremediated toxic-waste site face statistically-significant more harm, compared to children—born to the same mothers—but not conceived when their mothers were living within 3 km. Compared to their siblings, near-toxic- site children are 10 percent more likely to be diagnosed with a cognitive disability; 7 percent more likely to repeat a grade; more likely to have 6 percent (of the standard deviation) lower standardized test scores in grammar school; and 7 percent more likely to be suspended from school ,182.Because few people are likely aware of the preceding statistics, they may think children are merely “little adults,” with the same biological vulnerabilities as adults. Yet as Yet in spite of children’s medical vulnerabilities, many regulations do not adequately protect children from toxic chemicals , ionizinThis article performed a focused or limited-scope DUE for the Pasadena-toxic-site-remediation documents. One useful area of future DUE research would be to perform a focused DUE for Pasadena remediation documents that addresses other site contaminants besides chlorinated solvents such as PCE and TCE. For at least three reasons, it would be important to perform a DUE that addresses soil levels of site polyaromatic hydrocarbons (PAHs), such as benz(a)anthracene, benzo(a)pyrene, benzo(b)fluoranthene, and dibenzanthracene. One reason is that some of these PAHs far exceed allowable levels. A second reason is that some of them are genotoxic carcinogens, thus especially dangerous for children, including benz(a)anthracene, benzo(a)pyrene, benzo(b)fluoranthene chrysene, dibenzanthracene, and indenopyrene. A third reason to do future DUEs to address Pasadena-site PAHs is that many of them are semi-volatile organic compounds (SVOC) that are implicated in vapor intrusion, just as PCE and TCE are. Still another area of useful research would be to conduct a full-blown DUE for the Pasadena site, to determine whether other aspects of site-remediation documents exhibit similar problems with data usability.An additional area of important research would be to determine whether remediation documents for other toxic sites also have CSM data gaps that result in site-risk underestimation/determinate bias and therefore are likely to cause less protective cleanups. If other sites exhibit these data gaps and determinate bias, as the authors suspect, evidence from these other sites will help strengthen the preliminary conclusion of this article, namely, that routine, independent DUEs may be needed to help deter flawed PCE/TCE cleanups.implementing the highest data-quality/usability standards.Safe toxics cleanup may require enforcing the highest data-quality/usability standards.Safe toxics cleanup may require mandating routine, independent, data-quality/usability controls.Safe toxics cleanup may require prohibiting officials’ discretionary acceptance of flawed data.Safe toxics oversight may require The results of this Pasadena DUE case study suggest that without routine, independent DUEs, site-remediation documents may fail to have complete, usable data needed for reliable cleanup. Thus, if this article’s results are generalizable, they suggest several important principles:not implemented by states cannot guide state cleanups. Because both Pasadena city officials and the state DTSC approved the Pasadena toxic-site-remediation documents, despite their having numerous data gaps that jeopardize site safety, Principle 1 is important.Principle 1 is important because, as not enforced by state or federal authorities , data-quality/usability evaluation is Principle 4 is important, as many toxics’ regulators, like the California DTSC, have apparent authority to use “discretion” to ignore state/federal recommendations for data quality/usability. . If thisall risks from groundwater;airborne risks from windblown PM, carcinogenic vapors, and vapor intrusion;risks to many area receptors/victims, especially to onsite-resident and area children.The preceding data-usability evaluation (DUE) shows three major conceptual-site-model, scientific-data gaps in the Pasadena toxic-site-remediation documents, namely, data gaps regarding:Because these gaps appear to represent determinate biases that cause underreporting of site risk and needed remediation, Pasadena-remediation assessments fail this focused DUE and are likely to lead to flawed toxic-site cleanup. While many Pasadena residents could be hurt by deficient toxic-site remediation, the authors showed seven distinct reasons that children will be hurt most .The preceding results are important because they suggest one reason for flawed PCE and TCE remediation, namely, failure to perform routine, independent DUEs. If the results of this article prove to be generalizable, they suggest that routine, independent DUEs might help to (1) prevent PCE/TCE health harm; (2) provide an economical early-warning system for potentially flawed toxic-site cleanups; (3) deter scientific fraud in toxics remediation; (4) ensure more timely waste remediation; and (5) reduce legal and financial liability for toxic-waste sites.


The ability to catalyse a reaction acting on different substrates, known as “broad-specificity” or “multi-specificity”, and to catalyse different reactions at the same active site (“promiscuity”) are common features among the enzymes. These properties appear to go against the concept of extreme specificity of the catalytic action of enzymes and have been re-evaluated in terms of evolution and metabolic adaptation. This paper examines the potential usefulness of a differential inhibitory action in the study of the susceptibility to inhibition of multi-specific or promiscuous enzymes acting on different substrates. Aldose reductase is a multi-specific enzyme that catalyses the reduction of both aldoses and hydrophobic cytotoxic aldehydes and is used here as a concrete case to deal with the differential inhibition approach. In fact, numerous multispecific enzymes, able to catalyse in the active site the same transformation of different substrate, are knownOne aspect of the multi-specificity of enzymes, which to our knowledge has not been specifically addressed, concerns the feasibility, metabolic significance and usefulness of a differential inhibitory action directed at one or more, but not to all, of the reactions catalysed by a multi-specific enzyme. Our search for useful inhibitors of the multi-specific enzyme aldose reductase (AKR1B1) has led to our proposal of a new strategic approach for inhibiting the enzymeIn this paper, we propose that this inhibition strategy can be generalised as a fine approach to controlling enzyme activity, and we provide an overview of the conditions determining or favouring differential inhibition.A and B. For simplicity, we can consider that they are transformed with the same catk, into the products P and Q, respectively. In the presence of a classical competitive inhibitor (I), differential inhibition is predicted to occur in a situation (condition a) in which the MK for the two substrates (i.e. AK and BK) are significantly different. In a typical scenario, the inhibition test is performed separately on each of the two substrates present in the assay mixture. Here, by using the simple Michaelis and Menten steady-state kinetic equation, imposing a 5-fold difference in MK values between the two substrates (i.e. B/KAK = 5), and considering [I] = iK and [A] = [B] = B,K a differential inhibition of B with respect to A of approximately 19% will result. This value increases to approximately 25% for a B/KAK ratio of 10. Similarly, a differential inhibition may occur in a situation (condition b) in which the MK of the two substrates are similar in magnitude, but their concentrations are significantly different. Here, if [B] is fixed at the MK value while [A] is kept at 5-fold the MK value and [I] = iK, a differential inhibition of approximately 19% between the B transformation and the A transformation can be predicted. This value increases to 25% if [A] is raised to 10-fold MK, or to approximately 26% if [B] is decreased to a value of MK/2.The term “differential inhibition” refers to the inhibition of a multi-specific or a promiscuous enzyme acting on one or more specific substrates, while the transformation of other substrates remains unaffected or affected at a reduced extent. Differential inhibition is defined as the difference between the percent of inhibition of the reaction that is more sensitive to the inhibitor and that of other substrate transformation in the same reaction conditions. A differential inhibition may also occur for multi-specific and promiscuous enzymes in the presence of classical  inhibitors. This can depend on the conditions in which the enzyme’s susceptibility to inhibition is tested, and is linked to the kinetic parameters characterising the transformation of the two substrates. An intuitive example is that of a Michaelian enzyme acting on two competing substrates a) with B/KAK = 5, the relative equations describing products formation are:When both substrates are simultaneously present in the above conditions, the equation rate must consider, in addition to the inhibitor, the reciprocal influence exerted by the two substratesb  are inserted into B versus A, predicted to be 19% when each substrate is present alone, increases to 24%. In the same conditions as above, but with [B] = MK/2, an increase in differential inhibition from 26% when each substrate is present alone to 29% when the two substrates are simultaneously present is predicted.Thus, essentially due to the different reciprocal influence of the two substrates, an increase in differential inhibition from 19%, which is assumed when each substrate is present alone, to 24% is predicted. Similarly, when the parameters describing the condition in vivo physio-pathological situations the resulting differential inhibition may be a useful modulatory action for controlling enzyme function.Although the above conditions are simply imposed to provide an immediate result, many other different combinations of kinetic parameters and concentrations of substrates and inhibitor can occur, which may enhance or attenuate the apparent inhibitory differential effect. When the different reaction conditions for the two substrates represent possible In all the cases discussed above, we assume that the inhibitor we define as “classical” will intervene in the reaction irrespective of the substrate that the enzyme will transform. However, an inhibitory molecule may intervene differently in the transformation of two substrates when the interactions between the substrates and the active site are not the same. Thus, depending on the structural features of the substrates, different functional groups may be recruited and/or a different steric hindrance may result. This may well be the case for promiscuous enzymes, for which the two substrates undergo different reactions, thus enabling them to interact with different protein groups. In the most general case of multi-specific enzymes, which may or may not share the same pattern of functional groups that allow catalysis, the substrates may lead to a very specific range of interactions, particularly if they are significantly different in their structural features. This can then lead to a different interaction with the inhibitor. These conditions are the basis for a “mechanistic” generation of differential inhibition, in which the inhibitor is the active part of the phenomenon.DI) is a molecule that can interfere specifically with the transformation of one or more substrates while leaving the transformation of one or more other substrates free to occur. Differential inhibitors, which allow the enzyme to act on one of the possible substrates, may modulate the functions associated with multi-specific activityThus, the simplest definition of a “complete” intra-site differential inhibitor , which depends on the inhibitory features of the B transformation.This can be rationalised through the model shown in  derived , which ai/KiK′ ratio, which expresses the relative efficiency of the inhibitor in targeting the complex EB with respect to the free enzyme, clearly modulates the engagement of A in the inhibition process. Thus, the inhibitor progressively loses its character of DI, extending its action on the transformation of the substrate A.Computer simulations of appKi, appK′i), whose relative values may lead to apparent incongruities. For example, if the experimental data indicate a mixed type of inhibition for substrate B and an uncompetitive inhibition for substrate A, as in neohesperidin dihydrochalcone (NHDC), which has been assessed as an inhibitor of the reduction of L-idose (substrate B) and HNE (substrate A) catalysed by AKR1B120. The results of the analysis of substrate B shows that the inhibitor may target the free enzyme, and thus this should also result from the analysis of substrate A. However, the data suggest that the inhibitor targets only the EA complex. This apparently contradictory situation can be rationalised although not completely, in the following ways: (i) the measured apparent inhibitory constants can be regarded as true dissociation constants, in which the iK′ for substrate A is significantly lower than the iK measured for substrate B (which is not the case for NHDC); and/or (ii) the measured constants are indeed “apparent” and the ternary complexes EIA, which is generated by adding A to the EI complex, and EAI, which is generated by adding I to the EA complex, are kinetically different and only EIA is able to evolve into products. Thus, once the potential DI has been identified through individual substrate analysis, it will be necessary to proceed analysing the simultaneous presence of the competing substrates for a conclusive evaluation of the differential inhibitory ability of the selected molecule.The experimental measurements of the inhibition kinetic parameters that were independently conducted on the two substrates  refer to “apparent” inhibitory constants . This enzyme is an aldo-keto reductase able to catalyse the NADPH-dependent reduction of numerous aldehydic compounds. AKR1B1 is usually presented as the first enzyme of the so-called polyol pathway, in which glucose is reduced by the enzyme to sorbitol, which is in turn transformed into fructose through a NAD,15,In addition to its involvement in the polyol pathway, AKR1B1 has been recognised as fulfilling a detoxifying role, as it can efficiently reduce various toxic hydrophobic aldehydes, such as alkanals and alkenals generated through membrane lipid peroxidation as a result of oxidative stressin vitro discovery of selective and potent inhibitors of the enzyme. Thus, we propose a new strategy to approaching AKR1B1 inhibitionThe multifaceted activities of AKR1B1 have antagonistic effects that depend on the substrate undergoing reduction, and drug development from ARIs has been relatively poor when compared to the in vitro the differential inhibition of AKR1B1 mimic those occurring in a hyperglycaemic status, with the aldose substrates kept at the mM level, and the toxic aldehydes  or their glutathionyl-derivatives  kept at the µM level. D,L-glyceraldehyde, the most common substrate used in AKR1B1 inhibition studies, was also utilised as a substrate in differential inhibition studies. However, the evidence of incomplete inhibitory action exerted on the enzyme activity by aldose hemiacetals,Although a “complete” intra-site differential inhibitor as defined above has not as yet been envisaged for AKR1B1, molecules able to differentially inhibit aldoses reduction and/or GSHNE reduction versus HNE reduction have been proposed. The typical basic conditions to illustrate Thus, chemically synthesised compounds such as D-glyceramide or D-gluconamide were found to be inhibitors of the bovine lens enzymePhaseolus vulgaris, has been revealed to be a very promising source of ARDIs. After the bean was characterised in terms of classical AKR1B1 inhibitory activityNatural sources represent an almost limitless resource of biomolecules that can potentially affect the activity of enzymes, and this obviously also applies to the present inhibitory approach. Components exhibiting differential inhibitory ability were found in the fractionation of methanolic extracts of some edible vegetablesThe ability of enzymes to convert molecules that are structurally different through the same type of reaction, and the possibility of the same enzyme catalysing different reactions, may require the modulation of the enzyme activity depending on the substrate undergoing transformation. Thus, the “differential inhibitors” analysed in this work are the instruments that can specifically address the catalytic potential of multi-specific or promiscuous enzymes towards one of their possible multiple functions.Although no “complete” differential inhibitors of the multi-specific enzyme AKR1B1 have as yet been disclosed, knowledge of the interactive features and structural restraints a molecule needs to act as an ARDI is increasing. Studies conducted up to now on ARIs, in which ARDIs may have been disregarded, can be revisited, as an advantageous starting point to further this novel inhibition approach. Positive results for this enzyme target will also more generally confirm the potential of this intriguing inhibition approach for multi-specific or promiscuous enzymes.


A high number of children are referred to pediatric departments with a suspected allergic reaction to antibiotics. The prevalence of true allergy is considered to be significantly lower than shown from clinical history and symptoms alone. This study investigated the historical use of direct oral challenges at three specialist pediatric departments in Denmark.In this retrospective medical record review study, 141 children (69 boys and 72 girls) with a clinical history of suspected penicillin class allergy were investigated. A standardized questionnaire for drug allergy was completed in the beginning of the investigation, which also included a skin prick test (SPT), measurement of IgE to different types of penicillin, and a drug challenge (DC).Only four (2.8%) of the patients referred for further investigation in our study had a positive DC. We found no correlation between a positive DC, positive SPT or elevated specific IgE. None of the patients with a positive DC reacted with a rash alone prior to investigation.Allergy to penicillin in children is rare and probably overestimated. In children reacting to penicillin with a rash alone, our study indicated that the rash was probably not related to allergy and treatment should thus be continued. Only a small minority of children with suspected IgE-mediated allergy to penicillin is confirmed to be hypersensitive in direct oral challenge, which is the current reference standard. This study investigates the historical use of direct oral challenges at three specialist pediatric departments in Denmark. We speculated that most of the children referred with allergy to penicillin would show not to be.Many children are referred to pediatric departments with a suspected allergic reaction to antibiotics. The prevalence of true allergy is considered to be significantly lower than indicated from the clinical history and symptoms alone. Among children, allergic reactions have been reported with an incidence as high as 10% of all prescriptions for antibiotics . HoweverPenicillin is among the most commonly used antibiotics in children and thus often suspected to lead to allergic reactions in children . These mn = 52)), in vitro anti-penicillin and anti-cephalosporin specific IgE testing (n = 141), and measurements of total IgE level . The setup with SPT, DC and in vitro measurements of anti-penicillin and anti-cephalosporin specific IgE was in accordance with the European Network for Drug Allergy (ENDA) guidelines [This is a retrospective medical record review with no prospective interventional component. According to The Central Denmark Region Committee on Health Research Ethics, no formal ethics approval was required for this study. The study subjects were children followed in outpatient clinics at three Danish hospitals in Central Denmark Region, with a history of alleged allergy to a penicillin class antibiotic within the period from December 2007 to October 2011. In total, 141 children (69 boys and 72 girls) with an average age of 4.2 years  were included. The local guideline was a graded two-step direct oral challenge/drug challenge (DC), with two total hours of observation to confirm antibiotic allergy or current tolerance, along with completion of a standardized ENDA drug allergy questionnaire. There was variable use of skin prick testing (SPT) (only in a subgroup (idelines .SPT was performed in 52 (37%) of the children, primarily at one of the study hospitals (98%). SPT with benzylpenicillin, ampicillin and amoxicillin (1.25 mg/ml) was performed together with SPT with the culprit drug according to the patient history; histamine 10 mg/ml  and isotonic sodium chloride were used as controls. SPT was used on the volar forearm and read after 20 min. If the increase of the largest diameter of the rash was > 3 mm, the test was considered positive.The DC was performed as a graded oral challenge in all patients, including patients with positive SPT and/or positive specific IgE. Both a doctor and nurse were present all the time and the DC was performed following local anaphylaxis preparedness guidelines. The first dose was administered as one tenth of a normal treatment dose of penicillin/antibiotics . If no reaction was seen, a second full dose was administered 60 min after the first dose. The patients were observed for 2 h before leaving the hospital. Afterwards, the parents observed the child at home for 7 days and reported by telephone if late onset symptoms requiring evaluation occurred. At another study hospital, Amoxicillin was used in the DC. If the DC was positive, a new test with the culprit drug was performed. At the other two study hospitals, the culprit drug was used from the beginning. In both scenarios the oral challenge was stopped if a clinical reaction occurred.t test. Nominal statistical significance was set at a p-value < 0.05. All statistical analyses were performed using Sigmaplot 11.0, Systat Software.Hypothesis testing for continuous variables was done using the Symptoms leading to referral of children to the pediatric departments are shown in Table Most of the patients had several symptoms; the most common symptom was urticaria  followed by a small dotted macular itching exanthema  and a small dotted macular non-itching exanthema .The most common reason for starting treatment with antibiotics was sinusitis or otitis . Most children  had only reacted once to antibiotics before they were referred for a potential allergic reaction. Most often symptoms started within the first day of treatment  and persisted for less than 24 h after antibiotic treatment was stopped . Typically, no medication was given  to prevent the potential allergic reactions  followed by penicillin V or G  Positive SPT was seen in one patient  and one patient  had a positive specific IgE.Total IgE > 100 kU IgE/L was seen in 16%  of which one child had a positive DC. Furthermore, 32%  had a total IgE between 25 and 100 kU IgE/L (of which two children had a positive DC), and 52%  had a total IgE < 25 kU IgE/L (of which one child had a positive DC).Four patients (one boy and three girls aged 1.3 to 9.3 years) had a positive DC Table .Table 4Three reacted to amoxicillin and one to penicillin V; all four patients had a negative specific IgE and one had a negative SP (the three others were not tested).Of the four, one patient was referred with urticaria, one with angioedema, one with a combination of uritcaria and angioedema and the last one with a non-itching small dotted maculopapular exanthema and severe stomach pain Tables  and 4.All four were referred after their first allergic reaction; two had symptoms within the first day of antibiotic treatment and the other two within the first 2 days. Most of them (75%) had symptoms for less than 24 h. Two were not treated with any anti-allergic medication, and two were treated with antihistamine  and one six hours after a full dose. The last patient developed angioedema 20 min after 1/10 of a dose Table .2-agonist.One patient had a positive SPT to amoxicillin, but DC and specific IgE were negative. Total IgE was 8 kU IgE/L. In this patient, the index reaction consisted of urticaria, together with an itching small dotted macular exanthema and wheezing. The symptoms started within the first treatment day and lasted up to 48 h, although the patient was treated with oral steroids and inhalations with a βOne patient had a positive IgE  but negative DC as well as negative SPT to penicillin; the total IgE was 1122 kU IgE/L. The patient developed a non-itching small dotted macular exanthema within the first day of treatment. The symptoms lasted for less than 24 h and no treatment was needed.Only 2.8% of the patients referred to investigation of a potential allergic reaction to penicillin had clinically significant challenge reactions. This could indicate that allergy to penicillin in children has previously been overestimated as suggested by Esposito S et al.  We extenThe study increased focus on which symptoms predict a true allergic reaction to antibiotics. A macular exanthema rash alone, whether itching or not, was not likely to be associated with a true allergy to antibiotics and antibiotic treatment should thus not be ended or changed. This corresponds to the division of symptoms into low-risk or high-risk as proposed by Vyles D et al. We found no correlation between a positive DC, positive SPT or high specific IgE. In non-IgE allergic reactions the SPT as well as specific IgE has shown a low sensitivity , and in One of the strengths of the study is that all children in the same geographical region during a period of about 5 years were included, thus being representative for a Danish population of children referred for a potential allergic reaction to antibiotics. Another strength is that disregarding symptoms, results of IgE or SPT, all patients underwent a DC, which showed no correlations between a positive DC, positive SPT or elevated specific IgE.SPT was performed only in 37% of the children thus reducing the possibility to find a correlation with DC and specific IgE. We also used the culprit drug according to patient history; histamine and isotonic sodium chloride were used as controls. This could have reduced the sensitivity, as it would have been more correct to use the major determinant penicilloyl polylysine (Pre-Pen) or the minor determinants penicilloate and penilloate. However, Pre-pen was off the market from September 2004 to November 2009.The study have limitations due to the different setup strategies for investigating allergy to antibiotics, and thus not using the same algorithm for validation of the diagnosis of allergy to antibiotics including SPT, IgE measurement and DC. We know that allergic reactions are divided into immediate (IgE mediated reactions) and non-immediate (T-cell mediated) reactions . The oraFurther studies to investigate the use of especially specific IgE for penicillin are needed. Al though we found no evidence of a correlation between DC and IgE, we speculate that IgE will be helpful in reactions suggesting anaphylaxis, where a DC could be contraindicated.We concluded that allergy to penicillin in children is rare and probably overestimated. Only 2.8% of the patients referred due to suspected allergy in our study had a positive DC.In children reacting to antibiotics with a simple rash (itching or not) alone, our study indicates that the rash is most likely not related to allergy; antibiotic treatment should thus be continued.In diagnosing allergy, measuring specific IgE provided no useful information in our study. We suggest that all patients should undergo a three- to seven-day DC to increase the sensitivity of the diagnosis of non-IgE β-lactam allergy.


Donovanosis is a chronic, progressive, and indolent bacterial disease that affects the skin and mucous membranes of the genital and perigenital regions, often associated with sexual transmission, and with low infectivity. The malignant transformation of donovanosis lesions occurs exceptionally, as is usually seen in long-term ulcerations. Donovanosis is a chronic, progressive, and indolent bacterial disease that affects the skin and mucous membranes of the genital and perigenital regions, often associated with sexual transmission, and with low infectivity. The malignant transformation of donovanosis lesions occurs exceptionally, as is usually seen in long-term ulcerations.3It was first described by McLeod, in Calcutta, India, in 1882.Donovania granulomatis. The term donovanosis was proposed by Marmell and Santora in 1950 in honor of Donovan, and was accepted by the scientific community.In 1913, Aragão and Viana introduced intravenous emetic tartar treatment, the first effective drug in the treatment of donovanosis.Many synonyms for donovanosis can be found in the literature, such as phagedenic disease of the genitals, contagious granuloma, sclerosing granuloma, infectious granuloma, venereal granuloma, and the fifth venereal disease. These terms highlighted the clinical and epidemiological aspects of the disease.Much of what is known about donovanosis derived from studies carried out during the first half of the last century; few studies were conducted on the subject between the mid-1960s and 1990s. Since 1990, there has been an increase in scientific interest in this sexually transmitted infection (STI); much of this interest has arisen from the association between genital ulcers and HIV transmission.Calymmatobacterium granulomatis; however, they were very similar to Klebsiella spp., a similarity confirmed by Rake's studies based on serological crossing and, later, by ultrastructural differences.C. granulomatis was more than 90% similar to K. pneumoniae and K. rhinoscleromatis, proposing then the reclassification of the agent to Klebsiella granulomatis comb. nov., which remains to date.15The agent of donovanosis is a Gram-negative, facultative aerobic coccobacillus, which stains with greater intensity in the extremities than in the center, whether or not it is encapsulated, and which is intracytoplasmic and immobile. As a rounded cocus, it measures 0.02 µm to 0.2 µm; in the bacillary form, it measures 1.0 µm–2.5 µm in length.Although the disease has been described for more than a century, it is often overlooked due to its geographical distribution and low incidence. Therefore, it is not surprising that there are few published data on its incidence, even in endemic areas.Donovanosis is more common in blacks and individuals with poor hygiene, being endemic in tropical and subtropical countries such as Papua New Guinea, South Africa, India, Indonesia, Australia, the Caribbean, Argentina, French Guiana, and Brazil. However, it is likely that this distribution is more related to socioeconomic conditions and living conditions than to racial or geographical factors.Despite the worldwide decrease in cases of bacterial STIs and an increase in viral STIs, there is evidence to suggest a higher prevalence of unprotected sex after the introduction of antiviral therapy for HIV, which has been increasing the incidence of STIs in several industrialized and developing countries.Its incidence varies between the sexes in different studies, but there appears to be no gender predilection. It affects almost exclusively adults aged 20–40 years, a period of greater sexual activity; no congenital infections have been reported. However, there are reports of cases in infants and newborns, generally associated with contact with infected adults and not necessarily due to sexual abuse. There are few reports of perinatal donovanosis transmission, but the apparent predilection for otorhinolaryngological structures is noteworthy. Govender et al. reported two cases in which young children were diagnosed with donovanosis without a history of sexual abuse.The concomitance between donovanosis and STIs, including HIV, is well documented in the literature. As most cases of donovanosis presents clinically as a genital ulcer, the carrier has a 4.7 times increased risk of contracting HIV.As for its form of transmission, there is an interesting discussion in the literature if donovanosis is really an STI; there are two lines of thought regarding its transmission. The arguments that support the hypothesis of sexual transmission include:25Higher incidence in the age group of greater sexual activity.Lesions found in the internal genitalia, such as uterine cervix, without other manifestations.Presence of anal lesions associated with the practice of anal sex.Presence of external genital or near-genital lesions in most cases.Concomitant STI.Sexual contact with sex workers.High prevalence in sexual partners in some studies.In turn, arguments that these facts would not be sufficient to establish sexual transmission as definitive, supporting the non-sexual transmission of the disease, include:21Occurrence in children and adults without sexual activity.Relative rarity in sex workers.Unclear incubation period.Low rates of infection among sexual partners in some geographic areas.Occurrence of non-genital lesions in homosexuals and heterosexuals.C. granulomatis.Another argument contrary to sexual transmission was the isolation of microorganisms from the feces of a patient with donovanosis, morphologically similar to Despite the possibility of transmission through direct inoculation in some cases, donovanosis is currently considered primarily a STI, in which the main clinical manifestations occur in the genital region, with extragenital lesions occurring in 6% of cases. In favor of this argument is its higher incidence in sexually active groups, the predominant location in the genital areas (80%–100% of cases), and the frequent association of donovanosis, HIV, and other STIs in the same patient.29The incubation period is controversial and not well established in the literature; some studies indicate periods from two weeks to one month, others from 42 to 50 days, and experimental inoculations in human volunteers produced lesions after a period of 21 days.27The observation of the polymorphism of these manifestations led Jardim to propose a clinical classification that comprises the following forms:1. Genital and perigenital:1.1 Ulcerative;1.1.1 With hypertrophic edges;1.1.2 With flat edges.1.2. – Ulcerative-vegetative;1.3 Vegetative;1.4 Elephantiasis.2. Extragenital.Ulcerative forms may be lenticular in size, have everted edges, and present a mild inflammatory reaction. They grow slowly, becoming well defined, with a granular background, have a centrifugal and serpiginous character, and are covered with a thick and fetid exudate . They prElephantiasis manifestations (esthiomene) almost always occur after long-lasting ulcerated forms. They are consequent to lymphatic involvement 37Extra-genital locations  may be aIn patients with AIDS, due to the severe immunological depression that can occur, donovanosis can evolve with atypical clinical manifestations, with the appearance of new lesions or the expansion of those pre-existing, as well as persistence of bacteriological positivity, despite adequate treatment.40The possibility of depression of the cell-mediated immune response during pregnancy can favor infections by intracellular agents, such as donovanosis. The disease has a more aggressive course during pregnancy. In prenatal care, the hypothesis of donovanosis should be considered in cases of atypical lesions.The presence of genital ulcerations increases the risk of HIV transmission by up to 4.7 times after unprotected sexual exposure to a person infected with the virus. There are few studies on the relationship of the effects of HIV infection on donovanosis. However, as ulcers bleed very easily, it is understood that this disease is a large risk factor for the acquisition of HIV, a possibility that increases with the long duration of injuries. In areas where donovanosis is endemic, the prevalence of HIV has increased significantly since the beginning of its epidemic. This epidemiological aspect was observed in Durban, South Africa. This increase in HIV transmission has also been associated with the introduction of the syndromic approach for the treatment of STIs.42The diagnosis must be made taking into account the clinical aspect of the lesions, but it depends primarily on the demonstration of Donovan bodies in material obtained from the lesions, using cytological preparations and histological sections for confirmation.Direct microscopy is the fastest, most economical, and most reliable diagnostic method. The material should preferably be collected from areas of active granulation that do not show signs of secondary infection. The smears should be preferably collected from part of the fragment excised for anatomopathological examination, which should be pressed against a glass slide and then stained with Giemsa, Leishman or Wright staining .43FigureThe biopsy should be performed preferably on active lesions . The epidermis usually reveals acanthosis with a pseudoepitheliomatous aspect. The dermis shows an intense inflammatory infiltrate consisting mainly of lymphocytes, plasmocytes, histiocytes, and eventually a few eosinophils, with the presence of small neutrophilic abscesses in the epidermis and localized collections of neutrophils in the upper dermis. Histiocytes are usually bulky and vacuolar or cystic in appearance, and in their interior, it is possible to detect the inclusion bodies that are easily identifiable with Giemsa staining.C. granulomatis primes.21Culture of the etiological agent in the yolk sac of a chicken embryo is extremely difficult and is not routinely available due to its high cost, difficulty of execution, and high failure rate. Monolayer cell culture has been described using human monocytes, Hep-2 cells, and mouse peritoneal macrophages.The differential diagnosis of donovanosis lesions should be made with primary syphilis, flat condyloma, phagedenic chancroid, chronic herpetic ulcers, giant acuminate condyloma, lymphogranuloma venereum, and squamous cell carcinoma. It should also include diseases that can present ulcerative-granulomatous lesions, such as cutaneous tuberculosis, paracoccidioidomycosis, and pyoderma gangrenosum. Uncommon and infrequent diseases, such as Langerhans cell histiocytosis and bipolar aphthosis, may also require a differential diagnosis with donovanosis.27Intravenous emetic tartar, introduced by Aragon and Vianna in 1913, was the first effective drug in the treatment of donovanosis.,Currently, the recommended first-line therapy is azithromycin , two tablets of 500 mg a week for three weeks or until complete healing of the lesions.32Regardless of the therapeutic regimen adopted, the duration of treatment should be based primarily on complete healing of the lesions. In the event that scarring does not occur within six weeks, lesion biopsy must be performed to exclude the possibility of malignant transformation, particularly to squamous cell carcinoma. There is no relationship between the size of the lesions and response to treatment. In the rare cases in which relapse occurs after apparent clinical cure, treatment should preferably be repeated with a drug with an antimicrobial spectrum different from that used for primary infection and for a longer period than the first time. After treatment and complete healing of the lesions, the patient must be followed-up for at least one year, every two or three months.Among the most common complications are bleeding, genital lymphedema , deforming, and unsightly mutilations and scarring. The possibility of degeneration to squamous cell carcinoma should always be considered, particularly in cases with long evolution. If it is not diagnosed and treated in a timely manner, it can lead to metastases to nearby organs and put the patient's life at risk. Other complications that can affect these patients are scar adhesion of the scrotum to the penis, destruction of the penis body, and stenosis of the urethral, vaginal, and anal orifices. On exceptional occasions, especially in immunosuppressed patients, hematogenous dissemination and involvement of the liver, spleen, and bone may occur, with osteolytic lesions.None declared.Walter Belda Junior: Elaboration and writing of the manuscript.None declared.


The 3CLpro has attracted much interest due to its potential as a target for antiviral drugs. A system for growing high-quality crystals of native Southampton norovirus 3CLpro (SV3CP) has been established, allowing the ligand-free crystal structure to be determined to 1.3 Å in a tetrameric state. This also allowed crystal-based fragment screening to be performed with various compound libraries, ultimately to guide drug discovery for SV3CP. A total of 19 fragments were found to bind to the protease out of the 844 which were screened. Two of the hits were located at the active site of SV3CP and showed good inhibitory activity in kinetic assays. Another 5 were found at the enzyme’s putative RNA-binding site and a further 11 were located in the symmetric central cavity of the tetramer.Outbreaks of human epidemic nonbacterial gastroenteritis are mainly caused by noroviruses. Viral replication requires a 3C-like cysteine protease (3CL ORF2 anpro) was named because of its similarity to the picornavirus 3C protease. It is a cysteine protease which shows a typical chymotrypsin-like fold containing two domains: a β-barrel domain and a β-sheet domain separated by a groove where the active site is located  with an inhibitor bound. This compound consisted of part of the most rapidly cleaved substrate sequence (EFQLQ) with a Michael acceptor moiety linked to the P1 residue Gln. This is attacked by Cys139 and a covalently bound complex is formed. Interestingly, the His30 sidechain is pushed away by the inhibitor, which disrupts the catalytic triad.There is currently no clinically approved norovirus 3CLanalogue . Exampled assays . The aldive site . These c residue . Hussey pro in the crystals and two of them were located in the active site while another 5 were located at the enzyme’s putative RNA-binding site. A further 10 compounds were found to bind in the central cavity of this putative tetrameric form of the enzyme.Screening by mass-spectrometry for covalent inhibitors of SV3CP has been described by us previously . In this22.1plus, PACT premier, MIDAS and Morpheus from Molecular Dimensions . A TTP Labtech Mosquito crystal screening robot  was used to dispense 400 nl of the protein, at concentrations of 5 mg/ml and 10 mg/ml, with 400 nl of the corresponding well solution into each drop. High quality crystals were obtained in 0.2 M ammonium citrate and 12% (v/v) PEG3350 after approximately one week, although crystals kept appearing over the next 2–3 months prior to screening.Expression and purification of SV3CP was conducted using the method described by 2.2EDNA . Fine-sliced data were collected as guided by the strategy suggested by the program EDNA . Data weDNA  in 200 nl droplets containing 100 nl of the protein (4 mg/ml) and 100 nl of well solution . Since all of the fragments were dissolved in 100% dimethyl sulfoxide (DMSO), crystal stability in this solvent was first tested in the range (v/v) of 0%, 10%, 20%, 30% and 40%, and on soaking time scales of 1 h, 3 h and overnight. In order to make the experiment more efficient, the crystals were also tested with and without additional cryo-protectant for data collection. It was found that these crystals could survive in 40% DMSO for many hours and additional cryo-protection was not required.2.3.2TeXRank . All the crystals were then ranked using the program TeXRank  and posiTeXRank  and, dueTeXRank . Fragmen2.3.3XChemExplorer  and CueMol .The data produced were managed using Explorer  which ga , which was dissolved in DMSO to give final concentrations of 0.4, 0.9, 1.4, 1.9, 2.5 and 3.0 mM, in a 1:1 ratio and the absorbance at 405 nm was measured at 20 s intervals over a 3 min period, using a Nanodrop ND1000 spectrophotometer. The Ki values were determined using GraphPad Prism (www.graphpad.com).The protease  in a buffer containing 100 mM Tris, pH 8.5, and 5 mM β-mercaptoethanol was mixed with the fragment  for 20 min at RT. The solution was then mixed with the chromogenic substrate  which makes a catalytic triad with two residues from the N-terminal domain  between chains labelled A and D chains and likewise for chains labelled the B and C. This result indicates that higher order oligomers may possibly be formed by SV3CP dimers, such as the putative tetramer shown in pro. Indeed, in the structure of the Minerva virus enzyme (RCSB ID: 6b6i) the tetrameric assembly allows the C-terminal ends of two monomers  to extend into the active sites of adjacent monomers  across the dimer-dimer interface  at the C-terminal end of the protein. Since these residues are well-defined in the complex with a substrate analogue , their aositions  suggestiositions  confirmi3.2B-factors which are comparable with those of the protein moieties  were found to bind here, each on different sides of the catalytic cysteine . Five hi3.2.11 subsite where its carboxyl group is oriented towards S2 and S3. J01 forms several direct hydrogen bonds with the side chains of Gln110 and Arg112 and makes some additional hydrogen bonds mediated by a water molecule (β-hairpin (connecting strands β9 and β10) that is involved in substrate recognition and moves substantially upon binding of polypeptide substrate analogues  in the closed conformation, this must help to prevent the enzyme from adopting the 'open' conformation that can accommodate the substrate. The ligand –NH group (N1) is also within hydrogen bonding distance of the main chain carbonyl group of Thr134. The benzoic acid moiety of J01 makes many hydrophobic interactions with the active site residues including Pro136, Cys139 and Ala160. In contrast, the 5-methyl-2-thienyl group forms fewer contacts with the enzyme than the aromatic group since it points away from the active site towards a large solvent channel.Two non-covalently bound fragments were identified in the active site of the protease named as J01 and J02, as indicated by their omit maps a and c. molecule a and b. nalogues . However2 subsite without forming any hydrogen bonds (β-hairpin loop by π - π stacking and cation - π interactions. Interestingly, the guanidinium group of Arg112 has moved from its position in the other fragment complex to accommodate J02. Several hydrophobic interactions are formed between this fragment and Glu54 from the catalytic triad and Val114, and a number of contacts are made with a symmetry-related molecule.J02 resides on the other side of the long active site, where it occupies the Sen bonds c and d. Ki values of 0.37 mM and 0.34 mM, respectively. These values are typical of initial hits in crystallographic fragment-screening studies targetting catalytic- or allosteric-sites of enzymes (pro are highly relevant. Since J01 and J02 bind in the active site cleft and maintain the closed conformation of the hairpin, they are good candidates for developing further inhibitors and linking them into a new compound could also improve the bioactivity. A superposition of their binding modes on that of the covalently bound Michael acceptor inhibitor (1 and S2 subsites, respectively. J02 does not overlap with the P2 residue of the polypeptide inhibitor as well as J01 and the P1 residue do, since it appears to lie somewhere between the spatially adjacent S2 and S1′ subsites.In kinetic assays both J01 and J02 showed inhibitory activity against SV3CP with  enzymes  suggestinhibitor  demonstr3.2.250 of in the µM range (pro (PDB ID: 5fx5; pro (PDB ID: 2j92; pro. Inhibitors binding in the RNA binding site have the potential to inhibit noroviral replication and are therefore of interest as a separate class of drug.In addition to the protease activity, studies on viral 3C proteases suggested that they or their larger precursors can bind specifically to the 5′-terminal nucleotides of the viral RNA . The intµM range . The RNAµM range . StructuFragments J03-J06 were found to reside at this site and their contact residues are shown in 3.2.3The finding that the native crystals of the enzyme are formed by a tetrameric assembly of monomers is suggestive of a physiological role for the tetramer. We were also intrigued to find that the majority of the fragments binding to the protease (J07 - J17, 3.2.4Two of the fragments (J18 and J19, 4pro has been determined at 1.3 Å resolution in a crystal form that has allowed fragment-screening for novel inhibitors to be undertaken at similar resolutions. Two fragments were found to bind in the active site cleft of the protease. J01 and J02 bind in different subsites of the long active site (see β-hairpin linking strands β9 and β10. J01 occupies S1 and forms hydrophobic interactions with catalytic Cys139 while J02 occupies S2 and forms hydrophobic and π-π interactions with Glu54 and His30, which are also from the catalytic triad. Both J01 and J02 could potentially be developed into more potent norovirus protease inhibitors, however, a better ligand might ultimately be obtained by coupling them together, given that the distance between the closest two atoms is slightly less than 3.8 Å.The X-ray structure of the Southampton virus 3CLsite see  but bothin vivo activity, e.g. against mouse norvirus, which can be cultured, or in a suitable replicon assay. If such studies were to be successful, the highly symmetric nature of the binding site is something that could, in principle, be exploited in drug design.Some of the remaining fragments were found to interact with the protease at its putative RNA-binding site. Whilst these compounds are likely to have less effect on the protease activity than J01 and J02, which bind in the active site, RNA binding to the enzyme has been shown to cause non-competitive inhibition of the protease . Other fpro of coronavirus (e.g. 1 position of substrate. In very recent fragment screening of the SARS-CoV-2 protease, 23 active site hits were obtained which span the S3 to S1′ subsites of the enzyme, thus providing somewhat better coverage of the active site cleft than we have achieved with SV3CP (pro drug design is likely to have combined benefits for what are currently intractable and severe viral infections. These studies provide a rational basis on which compounds with improved potency can be designed by medicinal chemists.Given the recent COVID-19 pandemic, it is potentially useful to compare our results on SV3CP with the 3CLrus e.g. . The twoth SV3CP . Other Sth SV3CP . This reJingxu Guo: Conceptualization, Investigation, Methodology, Writing - original draft. Alice Douangamath: Methodology. Weixiao Song: Methodology, Investigation. Alun R. Coker: Supervision, Investigation. A.W. Edith Chan: Supervision, Investigation. Steve P. Wood: Supervision, Investigation. Jonathan B. Cooper: Supervision, Investigation, Writing - review & editing. Efrat Resnick: Investigation, Methodology. Nir London: Conceptualization, Methodology, Supervision. Frank von Delft: Conceptualization, Methodology, Supervision.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


The serine/threonine protein kinase AKT1 is a downstream target of the chemokine receptor 4 (CXCR4), and both proteins play a central role in the modulation of diverse cellular processes, including proliferation and cell survival. While in chronic myeloid leukemia (CML) the CXCR4 is downregulated, thereby promoting the mobilization of progenitor cells into blood, the receptor is highly expressed in breast cancer cells, favoring the migratory capacity of these cells. Recently, the LIM and SH3 domain protein 1 (LASP1) has been described as a novel CXCR4 binding partner and as a promoter of the PI3K/AKT pathway. In this study, we uncovered a direct binding of LASP1, phosphorylated at S146, to both CXCR4 and AKT1, as shown by immunoprecipitation assays, pull-down experiments, and immunohistochemistry data. In contrast, phosphorylation of LASP1 at Y171 abrogated these interactions, suggesting that both LASP1 phospho-forms interact. Finally, findings demonstrating different phosphorylation patterns of LASP1 in breast cancer and chronic myeloid leukemia may have implications for CXCR4 function and tyrosine kinase inhibitor treatment. LASP1 gene is located on chromosome 17q12 and encodes for a protein with an N-terminal LIM domain, followed by two nebulin-like repeats, a linker region with two phosphorylation sites at S146 and Y171, and a C-terminal SH3 domain, known to bind to proline-rich proteins like lipoma-preferred partner (LPP), zyxin, dynamin, vimentin, and zona occludens protein 2 (ZO2) -ATP for the times indicated.Constitutive active Lyn kinase and ABL kinase were purchased from Sigma-Aldrich and BPS Biosciences . Assay was performed with 0.01 µM kinase and 0.1 µM substrate (His-LASP1 and GST-CRKL) in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.25 mM DTT, 0.1 mM EGTA at 30 °C in the presence of 50 µM [mCXCR4-fwd: 5′-CCATGGAACCGATCAGTGTGmCXCR4-rev: 5′-TGAAGTAGATGGTGGGCAGGmCXCR7-fwd: 5′-GAAGCCCTGAGGTCACTTGGmCXCR7-rev: 5′-CACAGTGTCCACCACAATGCTM holder and kept in 190 µL of imaging buffer  containing 0.1% BSA. Then, 10 µL of 20 µM CXCL12 were carefully added into the solution to reach a final ligand concentration of 1 µM. The recording started at the same time as the ligand was added.Prior to transfection, HEK-293 cells were seeded on individual 24 mm glass coverslips placed in wells of a 6-well plate. The coverslips were pre-incubated with ploy-D-lysine for 30 min and washed once with PBS. After 6 h, the media was replaced with fresh media and the cells were transfected with 500 ng 3HA-CXCR4-CFP and 50 ng of Venus-LASP1 per well using the Effectene transfection reagent  according to the manufacturer’s instructions. Cells were used for confocal imaging 48 h after transfection. For the measurements, coverslips were mounted on an AttofluorThe experiments were performed using a Leica TCS SP8 confocal microscope . Images were taken using a 63× water objective . CFP was excited at 442 nm using a diode laser. CFP emission fluorescence intensity was detected from 455 to 510 nm. The YFP was excited with an argon laser at 514 nm and the emission fluorescence intensity was detected from 525 to 600 nm. The time series was recorded using sequential excitation, and images were taken at 3 s intervals for 20 min with a 512 × 512-pixel format, line average 1, frame average 1, and 400 Hz.4 K562 cells were fixed by adding 10% formalin into the culture medium for a final concentration of 5%. After 10 min incubation at 4 °C, cells were washed twice with cold PBS at 1000× g for 2 min at 4 °C, followed by centrifugation for 10 min at 140× g at RT to generate cytospins [w/v) paraformaldehyde in PBS on ice. Fixed cells were permeabilized with 0.1% (w/v) Triton X-100 in PBS and stained with monoclonal AKT1-Alexa Fluor 488  and affinity-purified polyclonal pLASP1-S146 PE-labelled antibody (diluted 1 µg/mL), as well as CXCR4-Alexa Fluor-488 antibody . PE-labeling of rabbit polyclonal pLASP1-S146 was performed with the Abcam R-Phycoerythrin conjugation kit , following the manufacturer’s instruction. Omnifocal analysis was done with a Biorevo BZ-9000 .For immunofluorescence microscopy, 3 × 10ytospins . MDAMB-2The 3D protein structure of the LASP1-SH3 domain was designed using PyMOL Molecular Graphics System, Version 2.0.4  open-source software.p < 0.05 .Paired two-tailed Student’s t-test for dependent groups was applied to determine the effects of LASP1 knockdown on AKT1-S473 phosphorylation. Results were considered significant at To confirm previous observations of the effect of LASP1 knockdown on AKT1 phosphorylation ,24,34, wBased on the previous data showing a LASP1-mediated PI3K/AKT1 activation ,24,34 anTo test for differences in LASP1 in MDAMB-231 and K562 cells, we measured the phosphorylation status of the protein in both cell lines. Interestingly, K562 cells mainly showed LASP1-Y171 phosphorylation, while MDAMB-231 cells demonstrated both phosphorylation at Y171 and S146 B. This dThese differences in LASP1 phosphorylation prompted us to test LASP1-AKT1 interaction with respect to different LASP1 phosphorylation states. Pulldown assays were performed with recombinant His-LASP1, either phosphorylated at S146 by constitutive active C-subunit of PKA, or at Y171 by active Lyn kinase, and lysates of HEK-293 cells transiently transfected with HA-tagged full-length AKT1. As depicted in +) like x+xPxxP or xPxxPx+ [It is known that the SH3 domain modulates a negatively charged cleft by aromatic amino acids, which interact with proline-rich sequences flanked by positively charged residues , as these cells harbor the chemokine receptor CXCR4 and share some common signaling pathways with leukemia stem cells [+Cxcr4flox/flox compared to CD11c-cre+Cxcr4+/+ mice (data not shown). After stimulation with the CXCR4 agonist CXCL12, cells were analyzed for AKT1 and LASP1 phosphorylation by Western blotting.Given the fact that CXCR4 is known to function as an upstream regulator of AKT1, combined with identification of LASP1 as a phosphorylation-dependent binding partner of CXCR4 and AKT1, we addressed if there was a “Yin-Yang” balance between serine and tyrosine phosphorylation of LASP1 and whether the above-mentioned in vitro observations were of any relevance em cells . SubsequIn a time-resolved analysis, a three-fold AKT1-S473 phosphorylation was observed within 2 min of CXCL12 stimulation, which declined back to basal levels after 10 min in control cells ), which It should be noted that we also checked for CXCR7 levels, as CXCL12 can also partially act through CXCR7 . HoweverTo further analyze the LASP1-CXCR4 interaction, we attempted to visualize the co-localization of both proteins in MDAMB-231 cells. However, immunofluorescence staining of CXCR4 failed due to low receptor expression and unspecific antibody recognition. It should be noted that we tested four different commercially available CXCR4 antibodies , among which only one showed a positive protein product of the correct molecular weight (not shown). We therefore generated CXCR4-CFP and Venus-LASP1 and transiently expressed the constructs in HEK-293 cells. Under basal conditions, a co-localization of both proteins at the cell membrane was seen . In paraTo substantiate our observations concerning the phospho-serine/phospho-tyrosine cross-regulation, we set out to test LASP1 phosphorylation in K562 cells before and after the inhibition of constitutively active tyrosine kinase BCR-ABL. The phosphorylation status of the adaptor protein CRKL A. HoweveAKT1 function is either regulated by direct inhibition/stimulation or by affecting subcellular localization. Activation mediates downstream responses, including cell survival, growth, proliferation, cell migration, and angiogenesis . This stIn the current study, we analyzed the newly identified direct phosphorylation-dependent binding of LASP1 to AKT1 and CXCR4, two major players in tumor progression. In view of the new results and in combination with earlier published data, we propose a model that explains the divergent roles of LASP1 and CXCR4 in solid breast cancer and in CML cells .In breast cancer, LASP1 is partially phosphorylated at S146 by PKA  or at Y1Based on our studies, we suggest that binding of pLASP1-S146 at the C-tail of CXCR4 stabilizes the receptor and sterically hinders the phosphorylation at multiple PKC serine phosphorylation sites important for receptor internalization, deactivation, and recycling back to the plasma membrane .i inhibits adenylyl cyclase/cAMP/PKA signaling and activates Src family tyrosine kinases, as seen in the transient dephosphorylation of LASP1 at the PKA-site (S146) and concomitant phosphorylation at Y171 (Upon stimulation by CXCL12, heterotrimeric G-protein is activated. Gα at Y171 . PhosphoA-site S16 and con at Y171 A, hence,Concomitantly, Gβγ release results in the activation of several pathways, i.e., PI3K/AKT , a key pIn contrast to breast cancer cells and other solid tumors , LASP1 iIn CML, chromosomal translocation results in an oncogenic BCR-ABL gene fusion that encodes for the constitutively active BCR-ABL fusion protein. As a result, hyper-phosphorylation of proteins is observed, among them the newly identified BCR-ABL substrate LASP1 . In contIn conclusion, we postulate a model for the divergent CXCR4-LASP1-AKT1 signaling in breast cancer and CML by differences in LASP1 phosphorylation. While pLASP1-S146 may contribute to CXCR4 stabilization in solid tumors, thereby promoting metastasis, LASP1-Y171 phosphorylation by BCR-ABL obviously hinders LASP1-CXCR4 binding and renders the chemokine receptor more sensitive to degradation in leukemic cells, thus favoring mobilization . Therefo


Escherichia coli, total coliform). The results of the analysis showed that the waters from both landfills were generally dominated by Ca, Mg, SO4, and HCO3 ions. Some of the major anions and cations in the water samples were above the South African National Standard (SANS241:2015) and World Health Organisation (WHO) permissible limits for drinking water. Majority of the boreholes had total dissolved solids and electrical conductivity values exceeding the SANS 241:2015 and WHO permissible limits. Piper trilinear plots for the two landfill sites showed that Ca(Mg)HCO3 water type predominates, but Ca(Mg)SO4 and Ca(Mg)Cl were also found. These water types were further confirmed with expanded Durov diagrams, indicating that that the boreholes represented a water type that is seldom found which is undergoing ion exchange, typical of sulphate contamination. From the SAR diagrams, boreholes in the northern landfill site had a high salinity hazard with only one borehole in the southern landfill site having a high salinity hazard. The geology was found to play a significant role in the distribution of contaminants into the groundwater systems in the study area. The study concluded that the northern landfill site had a poorer water quality in comparison to the southern landfill site based on the analysed physicochemical parameters. However, the southern landfill site showed significant microbial contamination, due to the elevated amount of E. coli and total coliform concentrations. The high permeability of the weathered dolerites in the northern landfill site might have enabled the percolation of contaminants into the groundwater resulting in the poorer water quality.This study assessed the groundwater quality around two municipal solid waste landfill sites, in the city of Bloemfontein, Free State Province, South Africa. The two landfill sites are located in two contrasting geological terrains, with both lacking some basic facilities found in a well-designed landfill. A total of eight groundwater samples were collected from pollution monitoring boreholes near the two landfill sites, with five samples representing the northern landfill site and three samples representing the southern landfill site. The samples were collected in the autumn and winter seasons to assess any possible seasonal variations. They were analysed for physicochemical , total dissolve solids (TDS), chemical oxygen demand (COD) and total organic carbon (TOC)) and microbiological parameters ( Water is regarded as an essential requirement of life which has been considered more as an economic resource than a social good . AccordiThe disposal of waste by landfilling has over the years proven to be one of the main culprits behind the contamination of groundwater resources . AccordiThere are a plethora of factors that need to be considered when selecting a site that will be suitable for locating a landfill site. Allistar and MacCarthy , suggestBloemfontein, a metropolitan city within the Free State Province of South Africa with an increasing population associated with people migrating from rural areas, resulting in an increase in a waste generation . GroundwIn this study, a comparison of the groundwater quality surrounding the two municipal landfill sites was conducted. It is important to investigate the impact of the leachate emanating from the unlined landfill on the groundwater chemistry. Physicochemical and microbiological parameters of the groundwater samples from the vicinity of the two landfill sites were analysed to assess the impact of leachate on the groundwater quality and determine whether there are any significant differences in the water chemistry obtained from the two landfill sites. The study area  compriseThe Bloemfontein northern landfill site is located in the northern margin of the city and is about 35 hectares in extent . The lanThe Bloemfontein southern landfill site is located in the southern margin of the city and is about 44 hectares in extent . The lanTo study the impact of leachate from the unlined municipal landfill sites, groundwater samples from the pollution monitoring boreholes in the vicinity of the two landfill sites (northern and southern) were collected . DuplicaFor the southern landfill site, a total of three sampling points “boreholes” consisting of boreholes SB04, SB08A and SB08B. The other monitoring boreholes  in the southern landfill site that are indicated in the map were not sampled as they were either damaged or collapsed. The water samples collected for physiochemical analysis were contained in tight-capped polyethene bottles, while the ones for microbiological analysis were in tight-capped glass bottles to avoid any contamination. They were kept in a cooler box containing ice before being transferred to the laboratory on the same day of collection for analysis. Water quality analyses  of the groundwater samples were carried out at the Institute for Groundwater Studies (University of the Free State) using ICP-MS, ion chromatography, and IDEXX (Colilert18) Quanti-TrayTM.Piper diagrams, expanded Durov diagrams and sodium adsorption ratio (SAR) diagrams were plotted using the WISH (Windows Interpretation for Hydrogeologists) version 3.02.191, a software program developed by Lukas Eelco from the Institute for Groundwater Studies, University of the Free State. This software has been used extensively by other researchers ,19. The The physicochemical and microbiological analyses of the water samples for autumn and winter for the two landfill sites are presented in Borehole samples from the northern landfill site had a mean TDS concentration of 2365 mg/L for the autumn samples and 1994.7 mg/L for the winter samples, with boreholes in the southern landfill site having mean TDS concentrations of 1005 mg/L for the autumn samples and 906 mg/L for the winter season, respectively . BoreholBorehole NB07 had the highest EC for the northern landfill site, and borehole SB04 had the highest EC value for the southern landfill site. The mean TOC and COD concentrations were also higher for boreholes in the northern landfill site in comparison to those in the southern landfill site. Boreholes from the northern landfill site had mean TOC of 27 mg/L for the autumn samples and 24 mg/L for the winter samples, while the samples from the southern landfill site had a mean of 6.9 mg/L for the autumn samples and 7.5 mg/L for the winter samples. COD concentrations also showed a similar trend with the other physicochemical parameters, with boreholes samples in the northern landfill site having a mean concentration of 142 mg/L for the autumn samples and 101 mg/L for the winter samples. The boreholes samples in the southern landfill site had a mean concentration of 24 mg/L for the autumn samples and 34 mg/L for the winter samples.Escherichia coli concentrations in comparison to the northern landfill site HCO3 vicinity over both seasons. These water samples are indicative of dominance in alkalinity and no dominant cation. Boreholes NB03A and NB03B also had similar chemistry and plotted in the Ca(Mg)SO4 vicinity in the two seasons. Borehole SB04 showed variation in the overall major ion chemistry over the two seasons. The borehole plotted in the mixed type vicinity in the autumn season and the Ca(Mg)Cl vicinity in the winter season. Borehole NB07 plotted in the Ca(Mg)Cl vicinity in the two seasons which indicated a dominance in the chloride anion and no dominant cation. The water samples from the two landfill sites showed variation in the overall major ion chemistry, as well as similarities. Boreholes NB06A and NB06B had similar chemistry and plotted in the Ca(Mg)HCOResults from the expanded Durovs shows the water samples from the northern landfill site plotted on the seldom found section, as well as the unpolluted water types based on minimum requirements suggested by the Department of Water Affairs requirements for water monitoring at waste management facilities. From a hydrogeochemical perspective, these water types are represented of water undergoing continuous changes in chemistry. Sodium Adsorption Ratio diagrams were constructed to illustrate the salinity hazard of the different groundwater samples based on their electrical conductivities . The SARAll the groundwater samples had a pH within the permissible limits as prescribed by WHO  and SANSGroundwater recharge through the dolerite dykes and fractures from rainfall that already contained elements in solution from the landfill could have facilitated more dissolution and caused considerable increases in TDS during autumn for the northern landfill site . RegardiAll the boreholes from the two landfill sites had EC values that exceeded the DWAF specifications for irrigation over both seasons. All the boreholes in the northern landfill site had EC values exceeding the recommended limits by WHO  and SANSThere are no given specifications for COD by both SANS241  and WHO E. coli in comparison to the northern landfill site over both seasons. All the boreholes from the two landfill sites had total coliform concentrations exceeding the recommended limit of less than 10 mg/L for drinking water as stipulated by SANS241 [According to Tripathi and Sharma , colifor SANS241  and WHO  SANS241 . E. coli concentrations over both seasons and were, therefore, within the recommended drinking water limits as stipulated by SANS 241 [E. coli concentrations, with borehole SB04 having the highest E. coli concentrations over both seasons. Findings by Elisante and Muzuka [E. coli counts were noted. The poor state of the borehole in the southern landfill site might have resulted in the borehole being prone to microbial contamination.Elisante and Muzuka , stipulaSANS 241  and WHO SANS 241 . All thed Muzuka  concludeIt is evident from the results and the discussion that the quality of the groundwater samples from the two landfill sites vary significantly, especially in terms of their physicochemical parameters and microbial quality. Although the two landfill sites both lack some required facilities of a modern landfill, are unlined, receive general “domestic” waste, located within the same geographical area and experience the same climatic conditions, the groundwater samples from the northern landfill site show more signs of contamination in comparison to the southern landfill site.k-values of between 0.4 and 0.6 m/day. Butler [k-values as most of the boreholes were drilled through dolerite. With reference to the southern landfill site, slug tests established that the permeability of the underlying siltstones and shale is low with a k-value of 2.76 × 10−3 m/day [When dolerite intrusions intrude into the surrounding rocks, geological structures, such as joints, form along the contact zone with the country rocks . Kale et. Butler  also fur−3 m/day . A study−3 m/day . A simil−3 m/day .The comparison of the groundwater quality from the two landfill sites, as well as the underlying geology highlights the importance of a detailed investigation that must be done prior to selecting a site suitable for a landfill HCO3 and Ca(Mg)Cl. The mixed hydrochemical facies point to constant changes of groundwater chemistry, due to the impact of the improperly designed landfills. This study assessed and compared the groundwater chemistry and quality from pollution monitoring boreholes in the vicinity of two landfill sites in Bloemfontein. A total of eight boreholes were sampled for water quality analysis. Three groundwater samples were taken from the southern landfill boreholes, while five boreholes samples were taken from the northern landfill boreholes. Hydrochemical facies analyses were determined using Piper and expanded Durov plots with Ca(Mg)SOThe groundwater usability status was unsafe, due to higher concentrations of Ca, Mg, Cl, Na, Pb, As, TDS and EC which were above the SANS241 and WHO drinking water quality standard and DWAF specification for irrigation, an indication that the groundwater was unfit for drinking, domestic, and irrigation purposes, due to the impact of the landfills on the groundwater quality. The analysed parameters were higher in the boreholes samples of the northern landfill site and much lower in the boreholes samples of the southern landfill site. Boreholes in the northern landfill site are of poorer quality with reference to the general physicochemical parameters, while boreholes in the southern landfill site had a poorer quality based on microbiological parameters. E. coli concentrations over both seasons, while boreholes in the southern landfill site had significant total coliform and E. coli concentrations over both seasons.Only one borehole in the southern landfill site had a high salinity hazard with the other two boreholes having a medium salinity hazard. There were no indications of significant metal concentrations in all sampled boreholes of both landfill sites. Comparison based on microbial parameters indicated that the boreholes in the northern landfill site had zero This study highlights that geology plays a significant role in the distribution of contaminants into groundwater systems. The presence of underlying dolerite in the northern landfill site resulted in the northern landfill site having a poorer water quality in comparison to the southern landfill site with respect to physicochemical parameters, while the poor state of the southern landfill boreholes made them more disposed to microbial contamination.