Coumarin-azasugar-benzyl conjugates as non-neurotoxic dual inhibitors of butyrylcholinesterase and cancer cell growth.

We have applied the copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction to prepare a library of ten coumarin-azasugar-benzyl conjugates and two phthalimide-azasugar-benzyl conjugates with potential anti-Alzheimer and anti-cancer properties. The compounds were evaluated as cholinesterase inhibitors, demonstrating a general preference, of up to 676-fold, for the inhibition of butyrylcholinesterase (BuChE) over acetylcholinesterase (AChE). Nine of the compounds behaved as stronger BuChE inhibitors than galantamine, one of the few drugs in clinical use against Alzheimer's disease. The most potent BuChE inhibitor (IC50 = 74 nM) was found to exhibit dual activities, as it also showed high activity (GI50 = 5.6 ± 1.1 µM) for inhibiting the growth of WiDr (colon cancer cells). In vitro studies on this dual-activity compound on Cerebellar Granule Neurons (CGNs) demonstrated that it displays no neurotoxicity.

Investigation of the enantioselectivity of acetylcholinesterase and butyrylcholinesterase upon inhibition by tacrine-iminosugar heterodimers.

The copper-catalysed azide-alkyne cycloaddition was applied to prepare three enantiomeric pairs of heterodimers containing a tacrine residue and a 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) or 1,4-dideoxy-1,4-imino-L-arabinitol (LAB) moiety held together via linkers of variable lengths containing a 1,2,3-triazole ring and 3, 4, or 7 CH2 groups. The heterodimers were tested as inhibitors of butyrylcholinesterase (BuChE) and acetylcholinesterase (AChE). The enantiomeric heterodimers with the longest linkers exhibited the highest inhibition potencies for AChE (IC50 = 9.7 nM and 11 nM) and BuChE (IC50 = 8.1 nM and 9.1 nM). AChE exhibited the highest enantioselectivity (ca. 4-fold). The enantiomeric pairs of the heterodimers were found to be inactive (GI50 > 100 µM), or to have weak antiproliferative properties (GI50 = 84-97 µM) against a panel of human cancer cells.

Conformationally Restricted Glycoconjugates Derived from Arylsulfonamides and Coumarins: New Families of Tumour-Associated Carbonic Anhydrase Inhibitors.

The involvement of carbonic anhydrases (CAs) in a myriad of biological events makes the development of new inhibitors of these metalloenzymes a hot topic in current Medicinal Chemistry. In particular, CA IX and XII are membrane-bound enzymes, responsible for tumour survival and chemoresistance. Herein, a bicyclic carbohydrate-based hydrophilic tail (imidazolidine-2-thione) has been appended to a CA-targeting pharmacophore (arylsulfonamide, coumarin) with the aim of studying the influence of the conformational restriction of the tail on the CA inhibition. For this purpose, the coupling of sulfonamido- or coumarin-based isothiocyanates with reducing 2-aminosugars, followed by the sequential acid-promoted intramolecular cyclization of the corresponding thiourea and dehydration reactions, afforded the corresponding bicyclic imidazoline-2-thiones in good overall yield. The effects of the carbohydrate configuration, the position of the sulfonamido motif on the aryl fragment, and the tether length and substitution pattern on the coumarin were analysed in the in vitro inhibition of human CAs. Regarding sulfonamido-based inhibitors, the best template turned out to be a d-galacto-configured carbohydrate residue, meta-substitution on the aryl moiety (9b), with Ki against CA XII within the low nM range (5.1 nM), and remarkable selectivity indexes (1531 for CA I and 181.9 for CA II); this provided an enhanced profile in terms of potency and selectivity compared to more flexible linear thioureas 1-4 and the drug acetazolamide (AAZ), used herein as a reference compound. For coumarins, the strongest activities were found for substituents devoid of steric hindrance (Me, Cl), and short linkages; derivatives 24h and 24a were found to be the most potent inhibitors against CA IX and XII, respectively (Ki = 6.8, 10.1 nM), and also endowed with outstanding selectivity (Ki > 100 µM against CA I, II, as off-target enzymes). Docking simulations were conducted on 9b and 24h to gain more insight into the key inhibitor-enzyme interactions.

Lactate dehydrogenase A inhibitors with a 2,8-dioxabicyclo[3.3.1]nonane scaffold: A contribution to molecular therapies for primary hyperoxalurias.

Human lactate dehydrogenase A (hLDHA) is one of the main enzymes involved in the pathway of oxalate synthesis in human liver and seems to contribute to the pathogenesis of disorders with endogenous oxalate overproduction, such as primary hyperoxaluria (PH), a rare life-threatening genetic disease. Recent published results on the knockdown of LDHA gene expression as a safe strategy to ameliorate oxalate build-up in PH patients are encouraging for an approach of hLDHA inhibition by small molecules as a potential pharmacological treatment. Thus, we now report on the synthesis and hLDHA inhibitory activity of a new family of compounds with 2,8-dioxabicyclo[3.3.1]nonane core (23-42), a series of twenty analogues to A-type proanthocyanidin natural products. Nine of them (25-27, 29-34) have shown IC50 values in the range of 8.7-26.7 µM, based on a UV spectrophotometric assay, where the hLDHA inhibition is measured according to the decrease in absorbance of the cofactor ß-NADH (340 nm). Compounds 25, 29, and 31 were the most active hLDHA inhibitors. In addition, the inhibitory activities of those nine compounds against the hLDHB isoform were also evaluated, finding that all of them were more selective inhibitors of hLDHA versus hLDHB. Among them, compounds 32 and 34 showed the highest selectivity. Moreover, the most active hLDHA inhibitors (25, 29, 31) were evaluated for their ability to decrease the oxalate production by hyperoxaluric mouse hepatocytes (PH1, PH2 and PH3) in vitro, and the relative oxalate output at 24 h was 16% and 19 % for compounds 25 and 31, respectively, in Hoga1-/- mouse primary hepatocyte cells (a model for PH3). These values improve those of the reference compound used (stiripentol). Compounds 25 and 31 have in common the presence of two hydroxyl groups at rings B and D and an electron-withdrawing group (NO2 or Br) at ring A, pointing to the structural features to be taken into account in future structural optimization.

Carbohydrate-derived bicyclic selenazolines as new dual inhibitors (cholinesterases/OGA) against Alzheimer's disease.

Concerned by the urgent need to explore new approaches for the treatment of Alzheimer's disease, we herein describe the synthesis and evaluation of new multitarget molecules. In particular, we have focused our attention on modulating the activity of cholinesterases (AChE, BuChE) in order to restore the levels of the neurotransmitter acetylcholine, and of O-GlcNAcase (OGA), which is associated with hyperphosphorylation of tau protein, in turn related to the formation of neurofibrillary tangles in the brain. Specifically, we considered the possibility of using carbohydrate-fused 1,3-selenazolines, decorated with a 2-alkylamino or 2-alkoxy moieties. On the one hand, the presence of a selenium atom might be useful in modulating the intrinsic oxidative stress in AD. On the other hand, such bicyclic structure might behave as a transition state analogue of OGA hydrolysis. Moreover, upon protonation, it could mimic the ammonium cation of acetylcholine. The lead compound, bearing a propylamino moiety on C-2 position of the selenazoline motif, proved to be a good candidate against AD; it turned out to be a strong inhibitor of BuChE (IC50 = 0.46 µM), the most prevalent cholinesterase in advanced disease stages, with a roughly 4.8 selectivity index in connection to AChE (IC50 = 2.2 µM). This compound exhibited a roughly 12-fold increase in activity compared to galantamine, one of the currently marketed drugs against AD, and a selective AChE inhibitor, and virtually the same activity as rivastigmine, a selective BuChE inhibitor. Furthermore, it was also endowed with a strong inhibitory activity against human OGA, within the nanomolar range (IC50 = 0.053 µM for hOGA, >100 µM for hHexB), and, thus, with an outstanding selectivity (IC50(hHexB)/IC50(hOGA) > 1887). The title compounds also exhibited an excellent selectivity against a panel of glycosidases and a negligible cytotoxicity against tumor and non-tumor cell lines. Docking simulations performed on the three target enzymes (AChE, BuChE, and OGA) revealed the key interactions to rationalize the biological data.

Translation and validation of the Portuguese version of the International Consultation on Incontinence Questionnaire (ICIQ) Bladder Diary.

INTRODUCTION AND HYPOTHESIS: The International Consultation on Incontinence Questionnaire (ICIQ) Bladder Diary (BD) is a standardized and validated diary, developed in the English language, designed to assess lower urinary tract symptoms (LUTS) in men and women. This study reports the translation, cross-cultural adaptation and validation process of this diary to the Portuguese language. METHODS: After translation and back-translation of the ICIQ-BD to Portuguese, 140 urologic patients were asked to fill in this 3-day diary as well as the Overactive Bladder questionnaire Short Form (OABqSF) and a questionnaire evaluating the difficulties in filling out the ICIQ-BD. A subset of 60 patients filled out a second diary with/without LUTS treatment in between (30 patients in each group). In addition, content validity, internal consistency, criterion and construct validity were tested. RESULTS: The Portuguese version of the ICIQ-BD showed adequate internal consistency (Cronbach's alpha of 0.78), and patients reported few difficulties in filling out this tool, answering most commonly 1 on a 1-6 scale of difficulty. Excellent test-retest reliability and responsiveness of the diary were observed when comparing the first diary to a second completed 2-6 weeks later. Criterion validity was also confirmed, given the good correlation with the OABqSF (Pearson's 0.386-0.447). Finally, construct validity was established through statistically significant concordance between data obtained in the BD with generally accepted theories. CONCLUSION: The present version of the ICIQ-BD is the first bladder diary successfully validated in the Portuguese language. It is a suitable and standardized tool for scientific research and diagnostic assessment of LUTS in adult men and women.

A hybrid of 1-deoxynojirimycin and benzotriazole induces preferential inhibition of butyrylcholinesterase (BuChE) over acetylcholinesterase (AChE).

The synthesis of four heterodimers in which the copper(I)-catalysed azide-alkyne cycloaddition was employed to connect a 1-deoxynojirimycin moiety with a benzotriazole scaffold is reported. The heterodimers were investigated as inhibitors against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). The heterodimers displayed preferential inhibition (> 9) of BuChE over AChE in the micromolar concentration range (IC50 = 7-50 µM). For the most potent inhibitor of BuChE, Cornish-Bowden plots were used, which demonstrated that it behaves as a mixed inhibitor. Modelling studies of the same inhibitor demonstrated that the benzotriazole and 1-deoxynojirimycin moiety is accommodated in the peripheral anionic site and catalytic anionic site, respectively, of AChE. The binding mode to BuChE was different as the benzotriazole moiety is accommodated in the catalytic anionic site.

2-Aminobenzoxazole-appended coumarins as potent and selective inhibitors of tumour-associated carbonic anhydrases.

We have carried out the design, synthesis, and evaluation of a small library of 2-aminobenzoxazole-appended coumarins as novel inhibitors of tumour-related CAs IX and XII. Substituents on C-3 and/or C-4 positions of the coumarin scaffold, and on the benzoxazole moiety, together with the length of the linker connecting both units were modified to obtain useful structure-activity relationships. CA inhibition studies revealed a good selectivity towards tumour-associated CAs IX and XII (Ki within the mid-nanomolar range in most of the cases) in comparison with CAs I, II, IV, and VII (Ki > 10 µM); CA IX was found to be slightly more sensitive towards structural changes. Docking calculations suggested that the coumarin scaffold might act as a prodrug, binding to the CAs in its hydrolysed form, which is in turn obtained due to the esterase activity of CAs. An increase of the tether length and of the substituents steric hindrance was found to be detrimental to in vitro antiproliferative activities. Incorporation of a chlorine atom on C-3 of the coumarin moiety achieved the strongest antiproliferative agent, with activities within the low micromolar range for the panel of tumour cell lines tested.

Squaramide-Tethered Sulfonamides and Coumarins: Synthesis, Inhibition of Tumor-Associated CAs IX and XII and Docking Simulations.

(1) Background: carbonic anhydrases (CAs) are attractive targets for the development of new anticancer therapies; in particular, CAs IX and XII isoforms are overexpressed in numerous tumors. (2) Methods: following the tail approach, we have appended a hydrophobic aromatic tail to a pharmacophore responsible for the CA inhibition (aryl sulfonamide, coumarin). As a linker, we have used squaramides, featured with strong hydrogen bond acceptor and donor capacities. (3) Results: Starting from easily accessible dimethyl squarate, the title compounds were successfully obtained as crystalline solids, avoiding the use of chromatographic purifications. Interesting and valuable SARs could be obtained upon modification of the length of the hydrocarbon chain, position of the sulfonamido moiety, distance of the aryl sulfonamide scaffold to the squaramide, stereoelectronic effects on the aromatic ring, as well as the number and type of substituents on C-3 and C-4 positions of the coumarin. (4) Conclusions: For sulfonamides, the best profile was achieved for the m-substituted derivative 11 (Ki = 29.4, 9.15 nM, CA IX and XII, respectively), with improved selectivity compared to acetazolamide, a standard drug. Coumarin derivatives afforded an outstanding selectivity (Ki > 10,000 nM for CA I, II); the lead compound (16c) was a strong CA IX and XII inhibitor (Ki = 19.2, 7.23 nM, respectively). Docking simulations revealed the key ligand-enzyme interactions.

Tuning the activity of iminosugars: novel N-alkylated deoxynojirimycin derivatives as strong BuChE inhibitors.

We have designed unprecedented cholinesterase inhibitors based on 1-deoxynojirimycin as potential anti-Alzheimer's agents. Compounds are comprised of three key structural motifs: the iminosugar, for interaction with cholinesterase catalytic anionic site (CAS); a hydrocarbon tether with variable lengths, and a fragment derived from 2-phenylethanol for promoting interactions with peripheral anionic site (PAS). Title compounds exhibited good selectivity towards BuChE, strongly depending on the substitution pattern and the length of the tether. The lead compounds were found to be strong mixed inhibitors of BuChE (IC50 = 1.8 and 1.9 µM). The presumptive binding mode of the lead compound was analysed using molecular docking simulations, revealing H-bond interactions with the catalytic subsite (His438) and CAS (Trp82 and Glu197) and van der Waals interactions with PAS (Thr284, Pro285, Asn289). They also lacked significant antiproliferative activity against tumour and non-tumour cells at 100 µM, making them promising new agents for tackling Alzheimer's disease through the cholinergic approach.

CKT0353, a novel microtubule targeting agent, overcomes paclitaxel induced resistance in cancer cells.

Microtubule targeting agents (MTAs) are extensively used in cancer treatment and many have achieved substantial clinical success. In recent years, targeting microtubules to inhibit cell division has become a widespread pharmaceutical approach for treatment of various cancer types. Nevertheless, the development of multidrug resistance (MDR) in cancer remains a major obstacle for successful application of these agents. Herein, we provided the evidence that CKT0353, α-branched α,ß-unsaturated ketone, possesses the capacity to successfully evade the MDR phenotype as an MTA. CKT0353 induced G2/M phase arrest, delayed cell division via spindle assembly checkpoint activation, disrupted the mitotic spindle formation and depolymerized microtubules in human breast, cervix, and colorectal carcinoma cells. Molecular docking analysis revealed that CKT0353 binds at the nocodazole binding domain of ß-tubulin. Furthermore, CKT0353 triggered apoptosis via caspase-dependent mechanism. In addition, P-glycoprotein overexpressing colorectal carcinoma cells showed higher sensitivity to this agent when compared to their sensitive counterpart, demonstrating the ability of CKT0353 to overcome this classic MDR mechanism involved in resistance to various MTAs. Taken together, these findings suggest that CKT0353 is an excellent candidate for further optimization as a therapeutic agent against tumors with MDR phenotype.

Assessment of indoor air conditions in households of Portuguese families with newborn children. Implementation of the HEALS IAQ checklist.

Conducting epidemiological and risk assessment research that considers the exposome concept, as in the case of HEALS project, requires the acquisition of higher dimension data sets of an increased complexity. In this context, new methods that provide accurate and interpretable data summary on relevant environmental factors are of major importance. In this work, a questionnaire was developed to collect harmonized data on potential pollutant sources to air in the indoor environment where children spend an important part of their early life. The questionnaire was designed in a user friendly checklist format to be filled out at the maternity in ten European cities. This paper presents and discusses the rationale for the selection of the questionnaire contents and the results obtained from its application in the households of 309 HEALS-enrolled families with babies recently born in Porto, Portugal. The tool was very effective in providing data on the putative air pollution sources in homes, with special focus on the bedroom of the newborns. The data collected is part of a wider effort to build the databases and risk assessment models of the HEALS project. The results of the analysis of the collected data suggest that, for the population under study, the main concerns on early life exposures at home can be related to emissions from the use of household solid fuels, indoor tobacco, household cleaning products, fragranced consumer products (e.g. air fresheners, incense and candles), moisture-related pathologies and traffic-related outdoor pollution. Furthermore, it is anticipated that the tool can be a valuable means to empower citizens to actively participate in the control of their own exposures at home. Within this context, the application of the checklist will also allow local stakeholders to identify buildings presenting most evident IAQ problems for sampling or intervention as well as to guide them in preparing evidence-based educational/awareness campaigns to promote public health through creating healthy households.

Inhibition of the mTOR pathway: A new mechanism of ß cell toxicity induced by tacrolimus.

The mechanisms of tacrolimus-induced ß cell toxicity are unknown. Tacrolimus (TAC) and rapamycin (Rapa) both bind to FK506-binding protein 12 (FKBP12). Also, both molecular structures are similar. Because of this similarity, we hypothesized that TAC can also inhibit the mTOR signalling, constituting a possible mechanism of ß cell toxicity. Thus, we studied the effect of TAC and Rapa over the mTOR pathway, v-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MafA), and insulin secretion and content in INS-1 ß cells treated with or without glucose and palmitate and in islets from lean or obese rats. TAC and Rapa inhibited the mTOR pathway as reflected by lower levels of phospho-mTOR, phospo-p70S6K, and phospo-S6. The effect of Rapa was larger than TAC. Both drugs reduced the levels of MafA, insulin secretion, and content although these effects were larger with TAC. The changes on MafA and insulin metabolism were observed in cells on glucose and palmitate, in obese animals, and were absent in cells on maintenance medium or in lean animals. In silico docking and immunoprecipitation experiments confirmed that TAC can form a stable noncovalent interaction with FKBP12-mTOR. Thus, the mTOR inhibition may be a mechanism contributing to the diabetogenic effect of TAC.

Phosphorylation of Mineralocorticoid Receptor Ligand Binding Domain Impairs Receptor Activation and Has a Dominant Negative Effect over Non-phosphorylated Receptors.

Post-translational modification of steroid receptors allows fine-tuning different properties of this family of proteins, including stability, activation, or interaction with co-regulators. Recently, a novel effect of phosphorylation on steroid receptor biology was described. Phosphorylation of human mineralocorticoid receptor (MR) on Ser-843, a residue placed on the ligand binding domain, lowers affinity for agonists, producing inhibition of gene transactivation. We now show that MR inhibition by phosphorylation occurs even at high agonist concentration, suggesting that phosphorylation may also impair coupling between ligand binding and receptor activation. Our results demonstrate that agonists are able to induce partial nuclear translocation of MR but fail to produce transactivation due at least in part to impaired co-activator recruitment. The inhibitory effect of phosphorylation on MR acts in a dominant-negative manner, effectively amplifying its functional effect on gene transactivation.

Stress test of a biological early warning system with zebrafish (Danio rerio).

The aim of this work was to develop a novel methodology to stress test the diagnostic capability of a video tracking system with zebrafish (Danio rerio), against two pre-established disturbances. Eight different treatments were tested varying the presence or absence of a toxicant (NaOCl) and two disturbances: the passing of a shadow (mimicking a predator) and entrapment of the fish. The concentration tested corresponded to a sublethal (1 % 24 h-LC50) and short term exposure (2 h). A total of 56 organisms were tested resulting in 112 diagnoses (before and after the contamination). A statistical model of diagnosis was developed using Self-organizing Map (SOM) and Correspondence Analysis (CA). Sensitivity, specificity, accuracy, false positive and false negative values were calculated to evaluate the diagnostic performance. The disturbances did not negatively affect the capability of the model. In the presence of at least one of these variables, the diagnostic performance was similar or even superior to the baseline results without disturbances. Furthermore, the system produced a large number of correct diagnoses, at an ecologically relevant concentration of exposure, in a non-invasive way.

Arthrotomy versus arthroscopy in the treatment of septic arthritis of the knee in adults: a randomized clinical trial.

PURPOSE: To evaluate the efficacy of treatment and functional recovery of patients diagnosed with septic arthritis of the knee submitted to two surgical techniques: conventional arthrotomy and arthroscopic approach for debridement. METHOD: In this prospective clinical study, all adult patients diagnosed with knee pyoarthritis in a 15-month period at a philanthropic hospital in Brazil were randomized into two groups: one group submitted to arthrotomy and the other to arthroscopy. The protocols of antibiotic therapy, physical therapy and postoperative assessment were standardized in both groups. Demographic, clinical, functional and laboratorial variables were compared between groups, with a minimum follow-up of 24 months. RESULTS: There was no difference in effectiveness of treatment in both groups, but 2 patients (18.2 %) of the arthrotomy group needed a new approach. The pain was higher in those undergoing treatment by arthrotomy, at 7 and 14 days postoperatively. The return to activities of daily living took an average of 5.7 days for both groups: 7.1 days for the arthrotomy group and 4.3 days for arthroscopy group. CONCLUSION: It was found that the treatment of knee septic arthritis by both techniques showed similar effectiveness in healing, but the arthroscopy procedure was better than arthrotomy because it has a lower reinfection rate and low initial inflammatory reaction. LEVEL OF EVIDENCE: Therapeutic studies, Level I.

Dual-stage triterpenoids from an African medicinal plant targeting the malaria parasite.

Sixteen triterpenoids (1-16), previously isolated from the aerial parts of the African medicinal plant Momordica balsamina or obtained by derivatization, were evaluated for their activity against liver stages of Plasmodium berghei, measuring the luminescence intensity in Huh-7 cells infected with a firefly luciferase-expressing P. berghei line, PbGFP-Luccon. Toxicity of compounds (1-16) was assessed on the same cell line through the fluorescence measurement of cell confluency. The highest activity was displayed by a derivative bearing two acetyl residues, karavoate B (7), which led to a dose-dependent decrease in the P. berghei infection rate, exhibiting a very significant activity at the lowest concentration employed (1 µM) and no toxicity towards the Huh-7 cells. It is noteworthy that, in previous studies, this compound was found to be a strong inhibitor of blood-stages of Plasmodium falciparum, thus displaying a dual-stage antimalarial activity.

Modelling the pH dependent retention and competitive adsorption of charged and ionizable solutes in mixed-mode and reversed-phase liquid chromatography.

This study investigated the influence of pH on the retention of solutes using a mixed-mode column with carboxyl (-COOH) groups acting as weak cation exchanger bonded to the terminal of C18 ligands (C18-WCX column) and a traditional reversed-phase C18 column. First, a model based on electrostatic theory was derived and successfully used to predict the retention of charged solutes (charged, and ionizable) as a function of mobile phase pH on a C18-WCX column. While the Horváth model predicts the pH-dependent retention of ionizable solutes in reversed-phase liquid chromatography (RPLC) solely based on solute ionization, the developed model incorporates the concept of surface potential generated on the surface of the stationary phase and its variation with pH. To comprehensively understand the adsorption process, adsorption isotherms for these solutes were individually acquired on the C18-WCX and reversed-phase C18 columns. The adsorption isotherms followed the Langmuir model for the uncharged solute and the electrostatically modified Langmuir model for charged solutes. The elution profiles for the single components were calculated from these isotherms using the equilibrium dispersion column model and were found to be in close agreement with the experimental elution profiles. To enable modelling of two-component cases involving charged solute(s), a competitive adsorption isotherm model based on electrostatic theory was derived. This model was later successfully used to calculate the elution profiles of two components for scenarios involving (a) a C18 Column: two charged solutes, (b) a C18 Column: one charged and one uncharged solute, and (c) a C18-WCX Column: two charged solutes. The strong alignment between the experimental and calculated elution profiles in all three scenarios validated the developed competitive adsorption model.

Biodegradation of PHB/PBAT films and isolation of novel PBAT biodegraders from soil microbiomes.

Polyhydroxyalkanoates (PHAs) are important candidates for replacing petroleum-based plastics. This transition is urgent for the development of a biobased economy and to protect human health and natural ecosystems. PHAs are biobased and biodegradable polyesters that when blended with other polymers, such as poly(butylene adipate-co-terephthalate) (PBAT), acquire remarkable improvements in their properties, which allow them to comply with the requirements of packaging applications. However, the biodegradation of such blends should be tested to evaluate the impact of those polymers in the environment. For instance, PBAT is a compostable aliphatic-aromatic copolyester, and its biodegradation in natural environments, such as soil, is poorly studied. In this work, we evaluated the biodegradation of a bilayer film composed of PHB and PBAT, by a soil microbiome. The bilayer film reached 47 ± 1 % mineralization in 180 days and PHB was no longer detected after this period. The increased crystallinity of the PBAT residue was a clear sign of biodegradation, indicating that the amorphous regions were preferentially biodegraded. Seven microorganisms were isolated, from which 4 were closely related to microorganisms already known as PHB degraders, but the other 3 species, closely related to Streptomyces coelicoflavus, Clonostachys rosea and Aspergillus insuetus, were found for the first time as PHB degraders. Most remarkably, two fungi closely related to Purpureocillium lilacinum and Aspergillus pseudodeflectus (99.83 % and 100 % identity by ITS sequencing) were isolated and identified as PBAT degraders. This is very interesting due to the rarity of isolating PBAT-degrading microorganisms. These results show that the bilayer film can be biodegraded in soil, at mesophilic temperatures, showing its potential to replace synthetic plastics in food packaging.

Estimating how contouring differences affect normal tissue complication probability modelling.

Background and purpose: Normal tissue complication probability (NTCP) models are developed from large retrospective datasets where automatic contouring is often used to contour the organs at risk. This study proposes a methodology to estimate how discrepancies between two sets of contours are reflected on NTCP model performance. We apply this methodology to heart contours within a dataset of non-small cell lung cancer (NSCLC) patients. Materials and methods: One of the contour sets is designated the ground truth and a dosimetric parameter derived from it is used to simulate outcomes via a predefined NTCP relationship. For each simulated outcome, the selected dosimetric parameters associated with each contour set are individually used to fit a toxicity model and their performance is compared. Our dataset comprised 605 stage IIA-IIIB NSCLC patients. Manual, deep learning, and atlas-based heart contours were available. Results: How contour differences were reflected in NTCP model performance depended on the slope of the predefined model, the dosimetric parameter utilized, and the size of the cohort. The impact of contour differences on NTCP model performance increased with steeper NTCP curves. In our dataset, parameters on the low range of the dose-volume histogram were more robust to contour differences. Conclusions: Our methodology can be used to estimate whether a given contouring model is fit for NTCP model development. For the heart in comparable datasets, average Dice should be at least as high as between our manual and deep learning contours for shallow NTCP relationships (88.5 ± 4.5 %) and higher for steep relationships.