Three epoxy-coated reinforcing steel (ECR) types removed from job sites, one shipped directly from the coater's plant, three commercial corrosion inhibitors, and one ECR plus a corrosion inhibitor were evaluated as reinforcing steel corrosion protection systems against chloride induced corrosion. The three corrosion inhibitors were calcium nitrite, an aqueous mixture of esters and amines, and a mixture of alcohol and amine. The ECR was tested in two groups, 0% and 1% coating damage. Corrosion protection performance was evaluated by the amount of visually observed blister surface area, for the ECR, and corroded surface area, for the tested corrosion inhibitors. Results of the ECR testing demonstrated that coating debondment and corrosion of ECR is directly related to the amount of damage present in the coating, as well as coating thickness. For the bare steel tested with and without corrosion inhibitors, the results showed that corrosion increases with increasing chloride concentrations. Corrosion inhibition characteristics were demonstrated only by the calcium nitrite corrosion inhibitor. A corrosion protection evaluation test was developed for concrete corrosion inhibitor admixtures. The test solution is a simulated concrete pore water. Corrosion is accelerated by evaluating the temperature to field conditions of 40 C. The test consists of a 7 day pretreatment period followed by a 90 day test period. The corrosive sodium chloride is added to the solution containing the bare or epoxy-coated reinforcing steel specimens after the 7 day pretreatment period. In addition, the solution is periodically saturated with oxygen. / Master of Science

<p dir="ltr">Cancer is the second leading cause of death worldwide, and there is a continued need for effective treatments to combat the disease. A key challenge in cancer therapy persists in the form of therapeutic resistance. While kinase inhibitors (KIs) have shown promise in treating cancer patients with dysregulated protein kinases, treatment failures are common, highlighting the urgent need to address this issue. Despite the approval of 80 protein kinase inhibitors by the United States Food and Drug Administration (FDA), and numerous others in clinical trials, the chemical space explored for protein kinase inhibitors remains limited. Most FDA-approved kinase inhibitors share common core moieties, such as indazole, quinoline, pyrazole, and pyrimidine, indicating a lack of diversification in drug development in this area.</p><p dir="ltr">Efforts to expand the chemical space have led to the identification of a novel 3<i>H</i>-pyrazolo-[4,3-<i>f</i>]quinoline core by the Sintim group. This scaffold can be efficiently synthesized through the Doebner–Povarov multicomponent reaction using readily available ketones, heteroaromatic aldehydes, and 5-aminoindazole. This multicomponent chemistry affords small molecules which inhibit disease-associated protein kinases with sub-nanomolar IC₅₀ values. Additionally, the scaffold presents a unique opportunity to tune for selectivity via judicious substitution patterns, allowing us to target numerous disease-driving kinases, such as FLT3, haspin, and CLK, with the use of simple multi-component chemistry.</p><p dir="ltr">From this work emerged lead amide-containing compound HSK205, which potently inhibits FLT3 and haspin and shows impressive potencies against FLT3-driven acute myeloid leukemia cell lines, with GI₅₀ values between 2 and 20 nM. Western blot analyses indicate that HSK205 inhibits the phosphorylation of FLT3 and histone H3 (substrate of haspin) in Molm-14 AML cells. Further exploration led to the discovery of lead CLK inhibitors, such as HSK1132 and HSK3110, which inhibit the growth of multiple myeloma cell lines <i>in vitro</i> with GI₅₀ values as low as 17 nM. Additionally, these compounds are orally bioavailable and reduce the growth of multiple myeloma RPMI-8226 xenograft model in mice by 69%.</p>

Target spot, caused by Corynespora cassiicola, is a common lower canopy disease of soybean in the southern United States. Given the recent resurgence of target spot and increasing reports of resistance to the quinone outside inhibitor (QoI) fungicide class within C. cassiicola, a survey of C. cassiicola from the Mississippi soybean production system was initiated in 2019 to determine the nature of its resistance mechanisms. A total of 819 monoconidial isolates were collected from 228 geographic field locations in 75 Mississippi counties. The molecular mechanism of resistance was determined using a PCR-RFLP analysis by comparing nucleotide sequences in the cytochrome b gene. The percentage of isolates containing the G143A substitution increased from 71.3% in 2016 to 93.5% in 2021. In all, 85.8% of the C. cassiicola isolates carried the G143A substitution. The EC50 values of QoI-resistant and -sensitive isolates to azoxystrobin varied significantly with QoI-sensitive isolates exhibiting lower EC50 values than QoI-resistant isolates. Moreover, results of fitness evaluations indicated that QoI-resistant isolates are more competitive than QoI-sensitive isolates and there were no fitness costs associated with QoI resistance in C. cassiicola. Additionally, the sensitivity of six C. cassiicola isolates to eight fungicide active ingredients in four fungicide classes were evaluated. Results indicated that three succinate dehydrogenase inhibitors benzovindiflupyr, fluxapyroxad, and pydiflumetofen were the most effective in inhibiting mycelial growth regardless of isolate phenotype followed by the methyl benzimidazole carbamate thiophanate-methyl, two demethylation inhibitors (DMI) difenoconazole and flutriafol, the QoI pyraclostrobin, and the DMI prothioconazole. Furthermore, the efficacy of seven commercial fungicides on target spot was evaluated in the greenhouse and field. Pydiflumetofen + difenoconazole, fluxapyroxad + pyraclostrobin, and thiophanate-methyl delayed disease progress and protected soybean yield, which indicated their effectiveness in managing target spot. Pydiflumetofen + difenoconazole also significantly reduced defoliation. Notably, fungicides applied at R3 were more effective in reducing disease severity and defoliation than additional growth stage timings. The current study revealed a reduction in C. cassiicola sensitivity to QoI fungicides and a shift to QoI-resistant populations exhibiting fitness advantages. Our findings provide pertinent information for growers as to which fungicides should be recommended to manage target spot.

Tesis por compendio / Goat milk is primarily destined for the production of fermented products, in particular cheese. Therefore, the control of antibiotic residues in milk is of great importance, since these could have negative repercussions on technological properties of the milk as well as on the health of consumers. In milk quality control programs, microbial inhibitor tests are widely applied to detect antibiotics during the screening stage. However, tests are non-specific and may be affected by substances other than antimicrobials which could inhibit the growth of the test micro-organism, causing false positive results. The aim of this thesis was to evaluate the interference, related to the presence of different contaminants in goat milk, on the response of microbial inhibitor tests commonly used in Spain to detect antibiotics (BRT MRL, Delvotest SP-NT MCS and Eclipse 100 tests). The influence of the physicochemical characteristics of goat milk on the false positive outcomes in microbial screening tests was also investigated. The suitability of microbial inhibitor tests for screening antibiotics in colostrum secretions was studied by analysing antibiotic-free colostrum and milk samples from forty-three Murciano-Granadina goats, collected every 12 hours during the first week post-partum. Microbial inhibitor tests were not suitable for the analysis of goat colostrum because they presented a high percentage of doubtful and positive results (up 37.2% in the 36 hours after partum). To evaluate the effect of caprine colostrum on the microbial test response, antimicrobial-free goat milk spiked with different concentrations of colostrum was analysed to calculate the inhibitory concentrations producing 5% of positive results. The highest interferences were obtained for the addition of colostrum from 12 to 24 hours post-partum and the colostrum concentrations producing 5% positive results were between 5.1 and 34.6%. The BRT MRL was the test the most affected. In another study, the interference of detergents and disinfectants used for the cleaning of milking equipment and milk storage tanks of dairy farms was investigated. Antimicrobial-free goat milk was spiked with eight concentrations of different cleaning products (5 acid, 5 alkaline, 5 domestic washing-up liquids, and 1 disinfectant) and analysed using microbial screening tests. The presence of acid detergent and disinfectant based on sodium hypochlorite in goat milk did not affect the microbial test response. However, alkaline detergents at concentrations ≥ 1 ml/l could lead to false positive results in microbial inhibitor tests (up to 16.7%) and from 4 ml/l on 100% positive results were obtained. Regarding the products used for home use, and those used on farms and small size dairies, washing-up liquid containing sodium laureth sulphate and ethanol had the greatest effects on microbial inhibitor tests, even starting from a relatively low concentration (1 ml/l). On the other hand, the presence of a relatively low concentration of detergents in goat milk (0.5 ml/l) slightly modified the detection capability of the microbial inhibitor tests for amoxicillin, ampicillin, benzylpenicillin, and cloxacillin, although the detection of these drugs at MRL (safe level) was not compromised. Antiparasitic agent residues in goat milk could be another possible cause of false positive results in microbial screening tests. An in vitro study to evaluate the effect of seven parasiticides commonly used in dairy goats was carried out. Further two studies, where albendazole and ivermectin were applied to two groups of dairy goats in lactation were performed. It should be noted that the parasiticide ivermectin is banned for the treatment of animals producing milk for human consumption, although its inclusion in this study was considered interesting to understand the potential effect of their residues in milk, in the event the practice was performed illegally. In the in vitro study, raw antibiotic-free milk from goats was spiked individually with eight different concentrations of albendazole, closantel, diclazuril, febendazole, levamisole, diazinon, and ivermectin. The microbial inhibitor test results showed a great variability according to the test and the drug under study. Of the tests considered, the BRT MRL test was the most sensitive to antiparasitic agents, with the lowest concentrations of antiparasitic agent causing 5, 10, and 50% of positive results. Generally, closantel and diazinon were the antiparasitic agents that produced higher interferences in all tests, since low concentrations already resulted in positive results, while only higher concentrations of diclazuril and ivermectin showed an inhibitory effect. To evaluate the effect of albendazole residues on the microbial inhibitor test response, eighteen healthy Murciano-Granadina goats in mid-lactation were treated with a single oral administration of the commercially available albendazole registered for dairy sheep (7.5 mg/kg b.w. of active compound) with a withdrawal period of 4 days for milk production in ovine. Albendazole and its metabolite residues in goat milk after under cascade treatment were not detected above MRL from the third day post-administration. However, a high occurrence of non-compliant results was obtained for the BRT MRL test during the first six days after treatment, suggesting that factors related to the albendazole application other than the drug concentration are able to affect the microbial inhibitor test response in some cases. Regarding the ivermectin study, twenty-eight Murciano-Granadina goats infested with Sarcoptes scabiei var. caprae were treated with a subcutaneous injection of ivermectin (200 μg/kg b.w.), with a second dose applied seven days after the first treatment. Drug residues in goat milk were recorded during the first fifteen days of the experiment with concentrations ranging from 8.13 to 24.25 ng/ml. In addition, all the microbial screening tests seem to be affected by the ivermectin treatment, with BRT MRL the most affected (20%) compared with Delvotest SP-NT MCS and Eclipse 100 (6.6 and 5.7%, respectively). These positive results cannot be associated with the ivermectin concentration in goat milk, as the concentrations measured were lower than the inhibitory concentrations as reported in a previous in vitro study for these microbial tests. Thus, as suggested by some authors, interferences could be related to changes or alterations caused by the application of the parasiticide agent or by the parasitic disease itself, which could affect the immune response of the animals favouring the presence of inhibitory substances in milk. The study of the effect of the goat milk composition on the specificity (rate of false positive results) of microbial inhibitor tests for screening antibiotics was also considered. Thus, individual goat milk samples (n=200) were analysed by microbial inhibitor tests using both visual and instrumental classification of the test results. The highest specificity values were obtained for the instrumental interpretation of the test results (94-99% vs 90-96%) due to the occurrence of samples with intermediate colorations (green-yellow, yellow-blue) making the visual classification more difficult and subjective. A relation was found between positive results in BRT MRL and Eclipse 100 tests and an elevated fat content in the goat milk. Positive outcomes in Eclipse 100 were associated with the butyric acid concentration in the milk. Further, the Delvotest SP-NT MCS test response was affected by elevated pH values, high lactoferrrin and myristoleic acid concentrations in the goat milk. This percentage of positive results could be minimized by a pre-treatment prior to microbial inhibitor test analysis, such as fat removal by centrifugation (3,100 g for 10 min at 4 ºC) and/or heating (80 ºC for 10 min). Undoubtedly, improvements on the specificity of the microbial inhibitor tests for screening antibiotics in goat milk are desirable to avoid the destruction of milk compliant for human due to the occurrence of false positive results. The related financial losses affect farmers and dairies. However, it should be noted that the presence of contaminants in goat milk could be avoided by applying good farming practices designed to ensure that milk is obtained from healthy animals under proper hygienic conditions so ensuring the food safety of goat milk and related dairy products. / Romero Rueda, T. (2015). Evaluation of false positive results in microbial inhibitor tests for screening antibiotics in goat milk [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/48552 / Compendio

Zebrafish thrombocytes exhibit characteristics of human platelets and megakaryocytes, making them valuable for studying megakaryopoiesis and thrombopoiesis. Using single-cell RNA sequencing, we analyzed gene expression in young and mature zebrafish thrombocytes. We identified 394 protein-coding genes unique to young thrombocytes, many corresponding with human orthologs, suggesting shared regulatory mechanisms in zebrafish and humans. We hypothesized knocking down these 394 genes should identify the novel regulatory genes that control thrombocyte maturation. To address this, we used the piggyback knockdown method to knock down these genes to study their biological functions in zebrafish thrombopoiesis. We first found the knockdown of nfe2, nfe2l1a, and nfe2l3 reduced both young and mature thrombocyte counts, confirming their role in thrombopoiesis. A comprehensive knockdown screening of the uniquely expressed genes in young thrombocytes identified 7 candidate genes associated with thrombopoiesis. We selected the spi1b gene for further mutant characterization, which revealed its critical role in young thrombocyte development, with homozygous mutations leading to embryonic lethality. Considering megakaryocyte properties in thrombocytes, we studied the potential for polyploidization in zebrafish thrombocytes. The inhibition of AURKA led to the development of polyploid thrombocytes resembling mammalian megakaryocytes, suggesting the retention of genetic programs for megakaryocyte development in zebrafish thrombocytes and providing insights into the evolutionary basis of thrombopoiesis. Thus, our study reveals critical gene expression patterns and regulatory factors in zebrafish thrombocyte development, offering insights into conserved mechanisms relevant to developmental biology and research in thrombosis and hemostasis disorder.

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No / PURPOSE: AQ4N is a novel bioreductive prodrug under clinical investigation. Preclinical evidence shows that AQ4N penetrates deeply within tumors and undergoes selective activation to form AQ4, a potent topoisomerase II inhibitor, in hypoxic regions of solid tumors. This proof-of-principle, phase I study evaluated the activation, hypoxic selectivity, and safety of AQ4N in patients with advanced solid tumors. EXPERIMENTAL DESIGN: Thirty-two patients with cancer (8 glioblastoma, 9 bladder, 8 head and neck, 6 breast, and 1 cervix) received a single 200 mg/m(2) dose of AQ4N before elective surgery. AQ4 and AQ4N levels in 95 tissues (tumor, healthy tissue) were assessed by liquid chromatography-tandem mass spectrometry. Tissue sections were also analyzed for AQ4 fluorescence using confocal microscopy, and for expression of the hypoxia-regulated glucose transporter, Glut-1. RESULTS: Activated AQ4 was detected in all tumor samples with highest levels present in glioblastoma (mean 1.2 microg/g) and head and neck (mean 0.65 microg/g) tumors; 22 of 32 patients had tumor AQ4 concentrations > or = 0.2 microg/g, levels previously shown to be active in preclinical studies. In 24 of 30 tumor samples, AQ4 was detected at higher concentrations than in adjacent normal tissue (tumor to normal ratio range 1.1-63.6); distant skin samples contained very low concentrations of AQ4 (mean 0.037 microg/g). Microscopic evaluation of tumor sections revealed that AQ4 colocalized within regions of Glut-1+ hypoxic cells. CONCLUSIONS: AQ4N was activated selectively in hypoxic regions in human solid tumors. Intratumoral concentrations of AQ4 exceeded those required for activity in animal models and support the evaluation of AQ4N as a novel tumor-targeting agent in future clinical studies.

Yes / Polysialic acid (polySia) is a linear polysaccharide comprised of N-acetylneuraminic acid residues and its over-expression in cancer cells has been correlated with poor clinical prognosis. An assay has been developed for quantitative analysis of cellular polySia expression. This was achieved by extracting and purifying released polySia from glycoproteins by mild acid hydrolysis and optimised organic extraction. The polySia was further hydrolysed into Sia monomers, followed by fluorescent labelling and quantitative analysis. The assay was qualified utilising endoneuraminidase-NF to remove polySia from the surface of C6-ST8SiaII cancer cells (EC50 = 2.13 ng/ml). The result was comparable to that obtained in a polySia-specific cellular ELISA assay. Furthermore, the assay proved suitable for evaluation of changes in polySia expression following treatment with a small molecule inhibitor of polysialylation. Given the importance of polySia in multiple disease states, notably cancer, this is a potentially vital tool with applications in the fields of drug discovery and glycobiology.

Kovalente Inhibition stellt einen effektiven Weg dar, die Verweildauer des Liganden innerhalb einer Bindetasche zu erhöhen. In dieser Arbeit wurden theoretische Methoden angewendet, um die Reaktivität und den nichtkovalenten Zustand vor der Reaktion zu modellieren. Im Rahmen einer Fallstudie zu Cathepsin K wurden nichtkovalente Modelle von kovalenten Inhibitoren generiert. Für verschiedene Komplexe aus Cathepsin K und einem kovalent gebundenem Liganden wurde der Zustand vor der Reaktion modelliert und dessen Stabilität im Rahmen einer klassischen MD-Simulation überprüft. Die Stabilität des Warheads in der Bindetasche hing hauptsächlich vom gewählten Protonierungszustand der katalytischen Aminosäuren ab. Für eine Reihe von Inhibitoren der ChlaDUB1 wurde ein Protokoll aus quantenmechanischen Rechnungen genutzt, um die Reaktivität verschiedener Warheads abzuschätzen. Die erhaltenen Aktivierungsenergien korrelierten mit experimentell bestimmten Raten zur Inaktivierung des Enzyms. Im Rahmen eines Wirkstoffdesign-Projektes zur Deubiquitinase USP28 wurden von unpublizierten Kristallstrukturen ausgehend erste Docking-Experimente durchgeführt. Es konnte gezeigt werden, dass ein literaturbekannter Inhibitor von USP28 mit einem Warhead so modifiziert werden kann, dass die reaktive Einheit in direkter Nachbarschaft zu einem Cystein positioniert wird. Für diese Warheads wurden ebenfalls quantenmechanische Rechnungen zur Bestimmung der Aktivierungsenergie durchgeführt. Um besser nachvollziehen zu können, warum bei einem Photoswitch-Inhibitor der Butyrylcholin-Esterase der cis-Zustand des Moleküls besser inhibiert als der trans-Zustand, wurde eine Docking-Studie des Zustandes vor der Reaktion durchgeführt. Es konnte ein qualitatives Modell aufgestellt werden, das zeigt, dass der trans-Zustand aufgrund seiner längeren Form mit wichtigen Aminosäuren am Eingang der Bindungstasche kollidiert. / Covalent inhibition is an effective way to increase the residence time of a ligand within the active site. In this work theoretical methods were used to model the reactivity and the noncovalent pre-reaction state. Noncovalent models of covalent inhibitors were generated as part of a case study of Cathepsin K. Several complexes of Cathepsin K and a covalently bound ligand were modeled in their state before the reaction, and their stability was assessed by classical molecular dynamics simulations. In most cases the warhead was positioned in close proximity to the catalytic unit, remaining there for up to several hundred nanoseconds. This stable positioning was largely dependent on the protonation state of the catalytic amino acids. To estimate the reactivity of a series of ChlaDUB1 inhibitors, a protocol of quantum mechanical calculations was adapted. The obtained activation energies correlated with experimentally obtained rate constants of enzyme inactivation. Using unpublished crystal structures, first design steps for the inhibition of the deubiquitinase USP28 were performed. Docking studies showed that modification of a literature-known inhibitor of USP28 with a warhead allowed to place this reactive unit close to a cysteine. Activation energies were also obtained for these structures via quantum mechanical calculations. To better rationalize the differences in inhibition between the cis- and trans-state of a photoswitch inhibitor of butyrylcholine esterase, a docking study of the noncovalent state was performed. The different ring conformers and stereochemical properties of the photoswitch were critical for a sensible model of the ligand. A qualitative model could be obtained which explains that the cis-isomer is more active than the trans-isomer due to a steric clash of the latter with amino acids at the entrance of the pocket.

NK/T-cell lymphoma (NKTCL) and T-cell non-Hodgkin lymphomas ( T-NHL) are highly aggressive lymphomas that lack rationally designed therapies and rely on repurposed chemotherapeutics from other hematological cancers. Histone deacetylases (HDACs) have been targeted in a range of malignancies, including T-cell lymphomas. This study represents exploratory findings of HDAC6 inhibition in NKTCL and T-NHL through a second-generation inhibitor NN-429. With nanomolar in vitro HDAC6 potency and high in vitro and in cellulo selectivity for HDAC6, NN-429 also exhibited long residence time and improved pharmacokinetic properties in contrast to older generation inhibitors. Following unique selective cytotoxicity towards T-NHL and NKTCL, NN-429 demonstrated a synergistic relationship with the clinical agent etoposide and potential synergies with doxorubicin, cytarabine, and SNS-032 in these disease models, opening an avenue for combination treatment strategies.