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IL-4 is known to present abnormal expression in thyroid tumors and SNPs in the IL-4 and its receptor IL-4R genes are associated to risk and mortality of various types of cancer. In order to evaluate their role in differentiated thyroid cancer (DTC), we investigated genetic frequencies of two IL-4 promoter SNPs (rs2070874 C>T, rs2243250 C>T) and four non-synonymous SNPs of the IL-4R gene (rs1805010 A>G, rs1805012 C>T, rs1805013 C>T, rs1801275 A>G) in 300 DTC patients matched to 300 controls. All patients were managed according to current guidelines and followed-up for a period of 12–252 months (69.20 ± 52.70 months). Although none of the six investigated SNPs showed association with risk of DTC, rs1805010 was associated with age of diagnosis and the SNPs rs1805012 and rs1801275 were associated to gender. Further, in-silico analysis showed that all these three SNPs were able to cause decreased stability of the protein. We were not able to demonstrate any other association to clinical features of aggressiveness or to patients’ prognosis. These findings indicate that although genetic variants in IL-4 and IL-4R do not influence the risk or outcome of DTC patients, their influence on the behavior of thyroid tumors deserves further investigation.

Malaria is a tropical disease that kills about half a million people around the world annually. Enzymatic reactions within pyrimidine biosynthesis have been proven to be essential for Plasmodium proliferation. Here we report on the essentiality of the second enzymatic step of the pyrimidine biosynthesis pathway, catalyzed by aspartate transcarbamoylase (ATC). Crystallization experiments using a double mutant ofPlasmodium falciparum ATC (PfATC) revealed the importance of the mutated residues for enzyme catalysis. Subsequently, this mutant was employed in protein interference assays (PIAs), which resulted in inhibition of parasite proliferation when parasites transfected with the double mutant were cultivated in medium lacking an excess of nutrients, including aspartate. Addition of 5 or 10 mg/L of aspartate to the minimal medium restored the parasites' normal growth rate. In vitro and whole-cell assays in the presence of the compound Torin 2 showed inhibition of specific activity and parasite growth, respectively. In silico analyses revealed the potential binding mode of Torin 2 to PfATC. Furthermore, a transgenic ATC-overexpressing cell line exhibited a 10-fold increased tolerance to Torin 2 compared with control cultures. Taken together, our results confirm the antimalarial activity of Torin 2, suggesting PfATC as a target of this drug and a promising target for the development of novel antimalarials.

In order to investigate the possible correlation between p53 and MDM2 co-expression with clinicopathological features of differentiated thyroid cancer (DTC) and its use as diagnostic and/or prognostic markers, we used immunohistochemistry to evaluate 317 thyroid samples including 208 DTC and 94 benign nodules, in addition to 15 normal tissues. MDM2 and p53 expression were highly associated (r = 0.7161; p 0.0001). The co-expression of p53-MDM2 was observed more frequently in malignant lesions (p 0.0001) and helped characterize follicular patterned lesions distinguishing FVPTC from FA (p 0.0001) and FVPTC from FTC (p 0.0001). In addition, p53-MDM2 co-expression was associated with characteristics of less aggressiveness. It was more frequent in patients ≤45 years old (p = 0.0035), with unique tumors (p = 0.0095), tumors2 cm (p 0.0001), tumors without extrathyroid invasion (p = 0.0425), without metastasis at evolution (p = 0.0179), and in patients evolving free of disease after treatment (p = 0.0485). We suggest that p53-MDM2 co-expression profile analysis might help establishing diagnostic and determining prognostic of DTC patients.

Introduction: Malaria is one of the most prevalent human infections worldwide with over 40% of the world's population living in malaria-endemic areas. In the absence of an effective vaccine, emergence of drug-resistant strains requires urgent drug development. Current methods applied to drug target validation, a crucial step in drug discovery, possess limitations in malaria. These constraints require the development of techniques capable of simplifying the validation of Plasmodial targets. Areas covered: The authors review the current state of the art in techniques used to validate drug targets in malaria, including our contribution - the protein interference assay (PIA) - as an additional tool in rapid in vivo target validation. Expert opinion: Each technique in this review has advantages and disadvantages, implying that future validation efforts should not focus on a single approach, but integrate multiple approaches. PIA is a significant addition to the current toolset of antimalarial validation. Validation of aspartate metabolism as a druggable pathway provided proof of concept of how oligomeric interfaces can be exploited to control specific activity in vivo. PIA has the potential to be applied not only to other enzymes/pathways of the malaria parasite but could, in principle, be extrapolated to other infectious diseases.

Aspartate transcarbamoylase catalyzes the second step of de-novo pyrimidine biosynthesis. As malarial parasites lack pyrimidine salvage machinery and rely on de-novo production for growth and proliferation, this pathway is a target for drug discovery. Previously, an apo crystal structure of aspartate transcarbamoylase from Plasmodium falciparum (PfATC) in its T-state has been reported. Here we present crystal structures of PfATC in the liganded R-state as well as in complex with the novel inhibitor, 2,3-napthalenediol, identified by high-throughput screening. Our data shows that 2,3-napthalediol binds in close proximity to the active site, implying an allosteric mechanism of inhibition. Furthermore, we report biophysical characterization of 2,3-napthalenediol. These data provide a promising starting point for structure based drug design targeting PfATC and malarial de-novo pyrimidine biosynthesis.

Investigate the clinical utility of serum interleukin dosages of IL-2, IL-2R, IL-4, IL-6, IL-6R, IL-8, IL-10 and IL-12 in the diagnosis and characterization of patients with DTC. In particular, verify ILs utility in the identification of individuals who are evolving disease-free or with the active disease.We evaluated 200 patients with malignant nodules (100 patients disease-free and 100 patients with recurrence/active disease); 60 benign nodules and 100 healthy controls, serum levels were assessed by ELISA.All ILs, but not IL-4, differentiated these three groups. We observed that IL-2, 2R and 10 serum concentrations were associated with thyroglobulin levels. Serum IL-2 was able to differentiate patients with active disease from the disease-free with a sensitivity of 98%, specificity of 58%, positive predictive value (PPV) of 70% and negative predictive value (NPV) of 97% (p=0.0007). IL-6R levels differentiated patients with active disease from the disease-free patients with 56% sensitivity, 63% specificity, PPV of 60% and NPV of 59% (p0.0001). IL-8 values also distinguished patients with active disease from the disease-free ones with sensitivity of 50%, specificity of 76%, PPV of 68% and NPV of 60% (p=0.0025); using IL-12, we obtained a sensitivity value of 73%, specificity of 66%, PPV of 68% and NPV of 71% (p0.0001). Furthermore, interleukin levels showed association with some tumor characteristics of aggressiveness.We suggest that the serum concentration of ILs may assist in the diagnosis and characterization of tumor malignancy helping identify patients with active disease who deserve closer medical attention.

Gastrointestinal stromal tumors (GISTs) are rare mesenchymal malignancies of the gastrointestinal tract. Most GISTs harbor a c-KIT (80%) or a PDGFR alpha (10%) mutation that leads to constitutive activation of the tyrosine kinase receptor. Response to treatment with tyrosine kinase inhibitors (TKIs) is dependent on mutational status of the tumor. The most common mutation in PDGFR alpha, D842V, is known to be imatinib resistant. Almost all other PDGFR alpha mutations are imatinib sensitive. We describe two patients with a PDGFR alpha exon 18 mutated GIST responding to treatment with TKIs. One of these patients has a p.M844_S847 deletion, not previously described in relation with TKI treatment response. Mutations in circulating tumor DNA were detectable with digital droplet PCR in serial plasma samples taken during treatment and correlated with treatment response of both patients. Computer 3D-modeling of the PDGFR alpha kinase domain of these two variants revealed no direct interference in imatinib or sunitinib binding and no effect in its activity in contrast to the reported structure of the imatinib resistant D842V mutation. An overview is given of the literature regarding the evidence of patients with different PDGFR alpha mutated GISTs on response to TKIs. The findings emphasize the use of mutational analysis in GIST to provide patients personalized treatment. Detection of mutations in plasma is feasible and can provide real-time information concerning treatment response. We suggest to register GIST patients with these uncommon mutations in a prospective international database to understand the tumor biology and obtain more evidence of such mutations to predict treatment response.

International journal of biological macromolecules 146, 212 - 221 (2020). doi:10.1016/j.ijbiomac.2019.12.252, The biosynthesis of pyoverdine, the major siderophore of Pseudomonas aeruginosa, is a well-organized process involving a discrete number of enzyme-catalyzed steps. The final step of this process involves the PvdP tyrosinase, which converts ferribactin into pyoverdine. Thus, inhibition of the PvdP tyrosinase activity provides an attractive strategy to interfere with siderophore synthesis to manage P. aeruginosa infections. Here, we report phenylthiourea as a non-competitive inhibitor of PvdP for which we solved a crystal structure in complex with PvdP. The crystal structure indicates that phenylthiourea binds to an allosteric binding site and thereby interferes with its tyrosinase activity. We further provide proofs that PvdP tyrosinase inhibition by phenylthiourea requires the C-terminal lid region. This provides opportunities to develop inhibitors that target the allosteric site, which seems to be confined to fluorescent pseudomonads, and not the tyrosinase active site. Furthermore, increases the chances to identify PvdP inhibitors that selectively interfere with siderophore synthesis., Published by Elsevier, New York, NY [u.a.]

Thiosulfate sulfurtransferase (TST, EC 2.8.1.1), also known as Rhodanese, is a mitochondrial enzyme which catalyzes the transfer of sulfur in several molecular pathways. After its initial identification as a cyanide detoxification enzyme, it was found that its functions also include sulfur metabolism, modification of iron-sulfur clusters and the reduction of antioxidants glutathione and thioredoxin. TST deficiency was shown to be strongly related to the pathophysiology of metabolic diseases including diabetes and obesity. This review summarizes research related to the enzymatic properties and functions of TST, to then explore the association between the effects of TST on mitochondria and development of diseases such as diabetes and obesity.

The appearance of multi‐drug resistant strains of malaria poses a major challenge to human health and validated drug targets are urgently required. To define a protein's function in vivo and thereby validate it as a drug target, highly specific tools are required that modify protein function with minimal cross‐reactivity. While modern genetic approaches often offer the desired level of target specificity, applying these techniques is frequently challenging—particularly in the most dangerous malaria parasite, Plasmodium falciparum. Our hypothesis is that such challenges can be addressed by incorporating mutant proteins within oligomeric protein complexes of the target organism in vivo. In this manuscript, we provide data to support our hypothesis by demonstrating that recombinant expression of mutant proteins within P. falciparum leverages the native protein oligomeric state to influence protein function in vivo, thereby providing a rapid validation of potential drug targets. Our data show that interference with aspartate metabolism in vivo leads to a significant hindrance in parasite survival and strongly suggest that enzymes integral to aspartate metabolism are promising targets for the discovery of novel antimalarials.

The appearance of multi-drug resistant strains of malaria poses a major challenge to human health and validated drug targets are urgently required. To define a protein's function in vivo and thereby validate it as a drug target, highly specific tools are required that modify protein function with minimal cross-reactivity. While modern genetic approaches often offer the desired level of target specificity, applying these techniques is frequently challenging-particularly in the most dangerous malaria parasite, Plasmodium falciparum. Our hypothesis is that such challenges can be addressed by incorporating mutant proteins within oligomeric protein complexes of the target organism in vivo. The present study provides data to support our hypothesis by demonstrating that recombinant expression of mutant proteins within P. falciparum leverages the native protein oligomeric state to influence protein function in vivo, thereby providing a rapid validation of potential drug targets. Our data show that interference with aspartate metabolism in vivo leads to a significant hindrance in parasite survival and strongly suggest that enzymes integral to aspartate metabolism are promising targets for the discovery of novel antimalarials.

Thede novopyrimidine-biosynthesis pathway ofPlasmodium falciparumis a promising target for antimalarial drug discovery. The parasite requires a supply of purines and pyrimidines for growth and proliferation and is unable to take up pyrimidines from the host. Direct (or indirect) inhibition ofde novopyrimidine biosynthesisviadihydroorotate dehydrogenase (PfDHODH), the fourth enzyme of the pathway, has already been shown to be lethal to the parasite. In the second step of the plasmodial pyrimidine-synthesis pathway, aspartate and carbamoyl phosphate are condensed toN-carbamoyl-L-aspartate and inorganic phosphate by aspartate transcarbamoylase (PfATC). In this paper, the 2.5 Å resolution crystal structure ofPfATC is reported. The space group of thePfATC crystals was determined to be monoclinicP21, with unit-cell parametersa= 87.0,b= 103.8,c= 87.1 Å, α = 90.0, β = 117.7, γ = 90.0°. The presentedPfATC model shares a high degree of homology with the catalytic domain ofEscherichia coliATC. There is as yet no evidence of the existence of a regulatory domain inPfATC. Similarly toE. coliATC,PfATC was modelled as a homotrimer in which each of the three active sites is formed at the oligomeric interface. Each active site comprises residues from two adjacent subunits in the trimer with a high degree of evolutional conservation. Here, the activity loss owing to mutagenesis of the key active-site residues is also described.

Objectives To investigate polymorphisms of IL-2 and IL-6R genes in patients with differentiated thyroid cancer (DTC), correlating them to previously obtained data on serum levels of IL-2 and IL-6R with DTC. Methods We evaluated the polymorphic inheritance of 300 malignant thyroid nodule patients and 300 healthy controls, including 200 patients with malignant nodules and 100 healthy controls previously investigated by ELISA for serum IL-2 and IL-6R levels. Results The genotypic distribution of IL-2 and IL-6R did not differ between cases and controls. Six haplotypes were generated for IL-6R SNPs, none of the haplotypes showed significance with the risk for thyroid cancer. Significant correlation was observed between all investigated polymorphisms and the serologic levels of the corresponding interleukins. Patients who had the IL-2 gene TT genotype of rs2069762 had higher serum levels of IL-2. Individuals with the CC genotype of rs2228145, the GG genotype of the rs4845622 and the AA genotype of the rs6684439 correlated with a higher serum production of IL-6R. Conclusion We demonstrated that IL-2 SNPs rs2069762, IL-6R rs2228145, IL-6R rs4845622 and IL-6R rs6684439 are associated with differences in the concentrations of the respective interleukins and these concentrations suggesting they may have a clinical utility distinguishing patient with active-disease.

Background: The validation of drug targets in malaria and other human diseases remains a highly difficult and laborious process. In the vast majority of cases, highly specific small molecule tools to inhibit a proteins function in vivo are simply not available. Additionally, the use of genetic tools in the analysis of malarial pathways is challenging. These issues result in difficulties in specifically modulating a hypothetical drug target’s function in vivo. Objective: The current “toolbox” of various methods and techniques to identify a protein’s function in vivo remains very limited and there is a pressing need for expansion. New approaches are urgently required to support target validation in the drug discovery process. Method: Oligomerisation is the natural assembly of multiple copies of a single protein into one object and this self-assembly is present in more than half of all protein structures. Thus, oligomerisation plays a central role in the generation of functional biomolecules. A key feature of oligomerisation is that the oligomeric interfaces between the individual parts of the final assembly are highly specific. However, these interfaces have not yet been systematically explored or exploited to dissect biochemical pathways in vivo. Results and Conclusion: This mini review will describe the current state of the antimalarial toolset as well as the potentially druggable malarial pathways. A specific focus is drawn to the initial efforts to exploit oligomerisation surfaces in drug target validation. As alternative to the conventional methods, Protein Interference Assay (PIA) can be used for specific distortion of the target protein function and pathway assessment in vivo.

Objective The transcriptional repressor DREAM is involved in thyroid-specific gene expression, thyroid enlargement and nodular development, but its clinical utility is still uncertain. In this study we aimed to investigate whether DREAM mRNA levels differ in different thyroid tumors and how this possible difference would allow the use of DREAM gene expression as molecular marker for diagnostic and/or prognosis purpose. Materials and methods We quantified DREAM gene mRNA levels and investigated its mutational status, relating its expression and genetic changes to diagnostic and prognostic features of 200 thyroid tumors, being 101 malignant [99 papillary thyroid carcinomas (PTC) and 2 anaplastic thyroid carcinomas] and 99 benign thyroid lesions [49 goiter and 50 follicular adenomas (FA)]. Results Levels of mRNA of DREAM gene were higher in benign (0.7909 ± 0.6274 AU) than in malignant (0.3373 ± 0.6274 AU) thyroid lesions (p < 0.0001). DREAM gene expression was able to identify malignancy with 66.7% sensitivity, 85.4% specificity, 84.2% positive predictive value (PPV), 68.7% negative predictive value (NPV), and 75.3% accuracy. DREAM mRNA levels were also useful distinguishing the follicular lesions FA and FVPTC with 70.2% sensitivity, 73.5% specificity, 78.5% PPV, 64.1% NPV, and 71.6% accuracy. However, DREAM gene expression was neither associated with clinical features of tumor aggressiveness, nor with recurrence or survival. Six different genetic changes in non-coding regions of DREAM gene were also found, not related to DREAM gene expression or tumor features. Conclusion We suggest that DREAM gene expression may help diagnose thyroid nodules, identifying malignancy and characterizing follicular-patterned thyroid lesions; however, it is not useful as a prognostic marker.

Many studies have provided observational data on the association of obesity and thyroid cancers, but only few of them propose mechanisms that would permit a better understanding of the causal molecular mechanisms of this association. Considering that there is an increasing incidence of both obesity and thyroid cancers, we need to summarize and link recent studies in order to characterize and understand the contribution of obesity-related factors that might affect thyroid cancer development and progression. Adipose tissue is involved in many vital processes, including insulin sensitivity, angiogenesis, regulation of energy balance, activation of the complement system, and responses such as inflammation. Although these processes have their own molecular pathways, they involve the same molecules through which obesity and adipose tissue might exert their roles in carcinogenesis, not only affecting MAPK and PI3K or even insulin pathways, but also recruiting local inflammatory responses that could result in disease formation and progression. This review describes five important issues that might explain the link between excessive weight and thyroid cancer: thyroid hormones, insulin resistance, adipokines, inflammation, and sexual hormones.

Malaria remains a major threat to human health, as strains resistant to current therapeutics are discovered. Efforts in finding new drug targets are hampered by the lack of sufficiently specific tools to provide target validation prior to initiating expensive drug discovery projects. Thus, new approaches that can rapidly enable drug target validation are of significant interest. In this manuscript we present the crystal structure of malate dehydrogenase from Plasmodium falciparum (PfMDH) at 2.4 Å resolution and structure-based mutagenic experiments interfering with the inter-oligomeric interactions of the enzyme. We report decreased thermal stability, significantly decreased specific activity and kinetic parameters of PfMDH mutants upon mutagenic disruption of either oligomeric interface. In contrast, stabilization of one of the interfaces resulted in increased thermal stability, increased substrate/cofactor affinity and hyperactivity of the enzyme towards malate production at sub-millimolar substrate concentrations. Furthermore, the presented data show that our designed PfMDH mutant could be used as specific inhibitor of the wild type PfMDH activity, as mutated PfMDH copies were shown to be able to self-incorporate into the native assembly upon introduction in vitro, yielding deactivated mutant:wild-type species. These data provide an insight into the role of oligomeric assembly in regulation of PfMDH activity and reveal that recombinant mutants could be used as probe tool for specific modification of the wild type PfMDH activity, thus offering the potential to validate its druggability in vivo without recourse to complex genetics or initial tool compounds. Such tool compounds often lack specificity between host or pathogen proteins (or are toxic in in vivo trials) and result in difficulties in assessing cause and effect-particularly in cases when the enzymes of interest possess close homologs within the human host. Furthermore, our oligomeric interference approach could be used in the future in order to assess druggability of other challenging human pathogen drug targets.

Background. We aimed to investigate a possible role of MAGE A3 and its associations with infiltrated immune cells in thyroid malignancy, analyzing their utility as a diagnostic and prognostic marker.Materials and Methods. We studied 195 malignant tissues: 154 PTCs and 41 FTCs; 102 benign tissues: 51 follicular adenomas and 51 goiter and 17 normal thyroid tissues. MAGE A3 and immune cell markers (CD4 and CD8) were evaluated using immunohistochemistry and compared with clinical pathological features.Results. The semiquantitative analysis and ACIS III analysis showed similar results. MAGE A3 was expressed in more malignant than in benign lesions (P<0.0001), also helping to discriminate follicular-patterned lesions. It was also higher in tumors in which there was extrathyroidal invasion (P=0.0206) and in patients with stage II disease (P=0.0107). MAGE A3+ tumors were more likely to present CD8+ TIL (P=0.0346), and these tumors were associated with less aggressive features, that is, extrathyroidal invasion and small size. There was a trend of MAGE A3+ CD8+ tumors to evolve free of disease.Conclusion. We demonstrated that MAGE A3 and CD8+ TIL infiltration may play an important role in malignant thyroid nodules, presenting an interesting perspective for new researches on DTC immunotherapy.