How Does Ethylenediaminetetraacetic Acid Irrigation Affect Biodentine? A Multimethod Ex Vivo Study.

The activity of biomaterials used during endodontic treatment can be affected by various factors. One of them is the chemical action of the irrigant that they are exposed to. The aim of this multimethod ex vivo study was to evaluate the influence of ethylenediaminetetraacetic acid (EDTA) on the surface appearance and chemical composition of Biodentine used in perforation repair. Twenty material specimens were prepared according to manufacturers' recommendations and divided into two setting-time-based groups, tested after 45 min (group A) and 24 h (group B) of setting. Material was irrigated with 17% EDTA solution with or without simultaneous ultrasonic activation. The surface characteristics and the chemical composition analysis of the Biodentine specimens were performed with the aid of a scanning electron microscope (SEM) and an energy dispersive spectroscopy (EDS) method, respectively. The volumetric loss of material was measured by dedicated digital software in an optical microscope. Statistical analysis was performed. The EDS study confirmed that after the rinsing protocol, the percentage content of elements differed between the groups. The EDTA rinse, whether ultrasonically activated or not, visibly affected the surface appearance and chemical composition of Biodentine. The specimens' surface subjected to irrigation was more irregular under SEM than in a control group. The US activation of the liquid amplified its impact on the tested material. The average volume loss in group A after 5 min irrigation was 3.98 µm3 for each µm2 of the chosen area and it increased up to 7.74 µm3/µm2 after the ultrasonic activation. In group B, indicated volume loss values were 6.30 and 11.70 µm3/µm2 for 5 min irrigation without and with US activation, respectively. Using a 20 min irrigation time and ultrasonic activation increased it up to 32.71 µm3/µm2. Each rinsing protocol involving irrigation with ethylenediaminetetraacetic acid modified the surface features and the chemical composition of the evaluated hydraulic tricalcium silicate cement. Further research is needed to indicate the possible impact of the observed changes on its long-term clinical performance.

The Utility of Intravenous Methylprednisolone as an Adjunct Treatment for Drug-Resistant Amiodarone-Induced Thyrotoxicosis.

BACKGROUND: Amiodarone-induced thyrotoxicosis (AIT) may pose treatment challenges. We present a series of patients in which we achieved the normalisation of free T3 (FT3) using intravenous methylprednisolone (ivMP) in AIT refractory to thiamazole and oral prednisone. Namely, in three males (aged 56, 50 and 64, all with a history of AF and/or a low ejection fraction), an addition of ivMP resulted in the normalisation of FT3, which allowed successful thyroidectomy. In another case of a 65-year-old man, we initially succeeded in the normalisation of FT3 using ivMP from FT4 > 7.77 ng/dL (0.93-1.7) to 2.41 ng/dL and in that of FT3 from 14.95 pg/mL (2-4.4) to 2.05 pg/mL), but four weeks after stopping ivMP, despite the continuation of thiamazole and prednisone, there was rebound thyrotoxicosis: FT4 > 7.77 ng/dL and FT3-5.46 pg/mL. Intravenous MP was restated leading to a decline in FT4 to 2.51 ng/dL and in FT3 to 1.92 pg/mL, thus allowing a successful thyroidectomy. Finally, in a 78-year-old man with AF, goitre, and AIT resistant to thiamazole, prednisone and lithium carbonate, we obtained a reduction in FT4 to 1.51 ng/dL and in FT3 to 3.17 pg/mL after seven pulses of ivMP. Oral prednisone was gradually reduced and successfully stopped about six months later. He remained on low-dose thiamazole (5 mg od). CONCLUSIONS: Pulse ivMP in addition to oral steroids may be a useful adjunct therapy either for the preparation of a thyroidectomy or as a treatment modality in drug-resistant AIT. Though a total cure is possible, there is a danger of a rebound worsening of thyrotoxicosis after premature discontinuation of ivMP.

Analysis of Reconstituted Tripartite Complex Supports Avidity-based Recruitment of Hsp70 by Substrate Bound J-domain Protein.

Hsp70 are ubiquitous, versatile molecular chaperones that cyclically interact with substrate protein(s). The initial step requires synergistic interaction of a substrate and a J-domain protein (JDP) cochaperone, via its J-domain, with Hsp70 to stimulate hydrolysis of its bound ATP. This hydrolysis drives conformational changes in Hsp70 that stabilize substrate binding. However, because of the transient nature of substrate and JDP interactions, this key step is not well understood. Here we leverage a well characterized Hsp70 system specialized for iron-sulfur cluster biogenesis, which like many systems, has a JDP that binds substrate on its own. Utilizing an ATPase-deficient Hsp70 variant, we isolated a Hsp70-JDP-substrate tripartite complex. Complex formation and stability depended on residues previously identified as essential for bipartite interactions: JDP-substrate, Hsp70-substrate and J-domain-Hsp70. Computational docking based on the established J-domain-Hsp70(ATP) interaction placed the substrate close to its predicted position in the peptide-binding cleft, with the JDP having the same architecture as when in a bipartite complex with substrate. Together, our results indicate that the structurally rigid JDP-substrate complex recruits Hsp70(ATP) via precise positioning of J-domain and substrate at their respective interaction sites - resulting in functionally high affinity (i.e., avidity). The exceptionally high avidity observed for this specialized system may be unusual because of the rigid architecture of its JDP and the additional JDP-Hsp70 interaction site uncovered in this study. However, functionally important avidity driven by JDP-substrate interactions is likely sufficient to explain synergistic ATPase stimulation and efficient substrate trapping in many Hsp70 systems.

Motives for using new psychoactive substances in three groups of Polish users: nightlife, marginalised and active on the In. / Motywy uzywania nowych substancji psychoaktywnych w trzech grupach uzytkowników: rekreacyjnych, zmarginalizowanych i aktywnych w internecie.

OBJECTIVES: The aim of this article is to present the motives for using new psychoactive substances (NPS) among users in Poland and to evaluate the relationships between motives and consumption of different types of NPS. METHODS: The fieldwork was conducted in four locations: in Warsaw, Krakow, Poznan, and Tricity. The study involved a total of 596 users of new psychoactive substances. Among them were: nightlife users, using NPS recreationally (N = 172), socially marginalised users (N = 86) and users active on the internet (N = 338). The technique used in the study was a self-filled questionnaire. RESULTS: As assessed by all respondents, enhancement of mood was the most frequently indicated motive for using NPS. Among nightlife users, the NPS were most often used for the purpose of having more fun at parties. Among the marginalised users, the most common motive for using the new psychoactive substances was the desire to get intoxicated, which is a motive belonging to the group of motives related to enhancement. In the group of people active on the Internet, the most common motives for using these substances were those related to expansion. CONCLUSIONS: Identifying motives for using new psychoactive substances may contribute to reducing the use of NPS. The recreational and marginalised users have different motives for using substances. The same conclusion applies to the use of the individual NPS. The motives of using them vary. Thus, preventive, educational and therapeutic programs should be judiciously adapted to the needs of the users as well as to the kind of substances they use.

A heterotypic assembly mechanism regulates CHIP E3 ligase activity.

CHIP (C-terminus of Hsc70-interacting protein) and its worm ortholog CHN-1 are E3 ubiquitin ligases that link the chaperone system with the ubiquitin-proteasome system (UPS). CHN-1 can cooperate with UFD-2, another E3 ligase, to accelerate ubiquitin chain formation; however, the basis for the high processivity of this E3s set has remained obscure. Here, we studied the molecular mechanism and function of the CHN-1-UFD-2 complex in Caenorhabditis elegans. Our data show that UFD-2 binding promotes the cooperation between CHN-1 and ubiquitin-conjugating E2 enzymes by stabilizing the CHN-1 U-box dimer. However, HSP70/HSP-1 chaperone outcompetes UFD-2 for CHN-1 binding, thereby promoting a shift to the autoinhibited CHN-1 state by acting on a conserved residue in its U-box domain. The interaction with UFD-2 enables CHN-1 to efficiently ubiquitylate and regulate S-adenosylhomocysteinase (AHCY-1), a key enzyme in the S-adenosylmethionine (SAM) regeneration cycle, which is essential for SAM-dependent methylation. Our results define the molecular mechanism underlying the synergistic cooperation of CHN-1 and UFD-2 in substrate ubiquitylation.

The Effect of Irrigation with Citric Acid on Biodentine Tricalcium Silicate-Based Cement: SEM-EDS In Vitro Study.

There are various factors that may interfere with the activity of biomaterials during endodontic therapy. One of them is the canal system irrigation procedure with different rinsing solutions performed after the placement of bioactive cements. The authors investigate the influence of citric acid, a chelating agent, on the surface and the chemical composition of Biodentine tricalcium silicate-based cement using a multimethod approach. Twenty samples were divided into two groups based on the material setting time. They were subjected to citric acid irrigation with or without ultrasonic activation for 5 and 20 min. The chemical analysis was made with energy dispersive spectroscopy (EDS). The visual assessment of Biodentine surface was carried out in scanning electron microscope (SEM). The volume of material loss during the procedure was measured with Keyence optic microscope and dedicated digital software. Statistical analysis was performed. The results of the study show that the irrigation with citric acid influenced the surface appearance of the material and changed its chemical composition in both investigated groups. The ultrasonic activation (US) of the liquid has also aggravated its impact. Further research is needed to assess if that fact may change the sealing properties of the material influencing the long-term clinical outcome.

Percutaneous Internal Ring Suturing (PIRS) - The Benefits of Laparoscopic Inguinal Hernia Repair.

INTRODUCTION: The laparoscopic inguinal hernia repair in children using the PIRS technique is a well-established method. However, there are still opinions that this method does not bring more benefits than open surgery. PURPOSE: The study aims to demonstrate the benefits of laparoscopic technique over conventional, open techniques. PATIENTS AND METHODS: We conducted a retrospective study that included children aged 0-18 treated using the PIRS technique in 2008-2016. The control group consisted of patients treated with the traditional, open method of inguinal hernia repair. A total of 276 children were qualified for laparoscopic surgery, and there were 274 patients in the control group. A full laparoscopic procedure was performed on 247 patients. Assessment of the pathology of the internal inguinal ring was done in all patients during the laparoscopic procedure. Intraoperatively 79 children had bilateral inguinal hernia diagnosed, 133 right-sided inguinal herniae, and 57 left-sided inguinal herniae. The occurrence of hernia was not confirmed in 7 children, whereas 53 patients had open contralateral patent processus vaginalis. In a case of contralateral patent processus vaginalis, the repair was performed using the PIRS method. RESULTS: The recurrence of the inguinal hernia was observed in 10 children in the laparoscopic group and in 5 cases in control group. The duration of the procedure was noted and compared to open inguinal hernia repair. There was a statistically shorter duration of the laparoscopic method. In the control group, there were 16 patients with a metachronous contralateral inguinal hernia. CONCLUSION: The laparoscopic inguinal hernia repair was associated with a better assessment of hernia pathology, shorter operative time, and lower risk of contralateral hernia repair.

Electrospray-Induced Mass Spectrometry Is Not Suitable for Determination of Peptidic Cu(II) Complexes.

The toolset of mass spectrometry (MS) is still expanding, and the number of metal ion complexes researched this way is growing. The Cu(II) ion forms particularly strong peptide complexes of biological interest which are frequent objects of MS studies, but quantitative aspects of some reported results are at odds with those of experiments performed in solution. Cu(II) complexes are usually characterized by fast ligand exchange rates, despite their high affinity, and we speculated that such kinetic lability could be responsible for the observed discrepancies. In order to resolve this issue, we selected peptides belonging to the ATCUN family characterized with high and thoroughly determined Cu(II) binding constants and re-estimated them using two ESI-MS techniques: standard conditions in combination with serial dilution experiments and very mild conditions for competition experiments. The sample acidification, which accompanies the electrospray formation, was simulated with the pH-jump stopped-flow technique. Our results indicate that ESI-MS should not be used for quantitative studies of Cu(II)-peptide complexes because the electrospray formation process compromises the entropic contribution to the complex stability, yielding underestimations of complex stability constants.

The Role of Nrf2 Transcription Factor and Sp1-Nrf2 Protein Complex in Glutamine Transporter SN1 Regulation in Mouse Cortical Astrocytes Exposed to Ammonia.

Ammonia toxicity in the brain primarily affects astrocytes via a mechanism in which oxidative stress (OS), is coupled to the imbalance between glutamatergic and GABAergic transmission. Ammonia also downregulates the astrocytic N system transporter SN1 that controls glutamine supply from astrocytes to neurons for the replenishment of both neurotransmitters. Here, we tested the hypothesis that activation of Nrf2 is the process that links ammonia-induced OS formation in astrocytes to downregulation and inactivation of SN1 and that it may involve the formation of a complex between Nrf2 and Sp1. Treatment of cultured cortical mouse astrocytes with ammonia (5 mM NH4Cl for 24 h) evoked Nrf2 nuclear translocation, increased its activity in a p38 MAPK pathway-dependent manner, and enhanced Nrf2 binding to Slc38a3 promoter. Nrf2 silencing increased SN1 mRNA and protein level without influencing astrocytic [3H]glutamine transport. Ammonia decreased SN1 expression in Nrf2 siRNA treated astrocytes and reduced [3H]glutamine uptake. In addition, while Nrf2 formed a complex with Sp1 in ammonia-treated astrocytes less efficiently than in control cells, treatment of astrocytes with hybrid-mode inactivated Sp1-Nrf2 complex (Nrf2 silencing + pharmacological inhibition of Sp1) did not affect SN1 protein level in ammonia-treated astrocytes. In summary, the results document that SN1 transporter dysregulation by ammonia in astrocytes involves activation of Nrf2 but does not require the formation of the Sp1-Nrf2 complex.

Negative consequences of novel psychoactive substances use among the Polish users. / Negatywne konsekwencje wynikajace z przyjmowania nowych substancji psychoaktywnych przez polskich uzytkowników.

OBJECTIVES: The use of the novel psychoactive substances (NPS) is a relatively new phenomenon on the Polish drug scene. At the same time, it is rapidly growing group of substances. The aim of the article is to present the negative consequences to physical, mental and social health due to novel psychoactive substances use. METHODS: Fieldwork was conducted in 2016 in four locations: Warsaw, Krakow, Poznan, and Tri-City. A total of 596 users of novel psychoactive substances participated in the study. Among them were: nightlife users (N = 172), marginalised users (N = 86) and users active on the internet (N = 338). A self-administered questionnaire was used. RESULTS: The substances from the stimulant/empathogen/nootropics group were the most commonly used among nightlife users, marginalised users and users active on the internet. The herbal blends were less popular, and the synthetic cannabinoids appeared on the third position. The acute side effects were most common consequences. Prevalence of medium- and long-termproblems withmentaland physicalhealth as well as social problems was at a similar level. All types of negative consequences were most prevalent among marginalised users. CONCLUSIONS: Novel psychoactive substances users experience several negative consequences. This poses a challenge to the treatment services that must answer with adequate form of help. This is not easy due to diagnostic difficulties as the effects of NPS use are often indistinguishable to the symptoms of the traditional drugs. Thus, there is a need for further research, which would allow to observe and closely monitor the problem.

Patients' and professionals' beliefs about the impact of social stigmatization on treatment of gambling-related disorders. / Przekonania pacjentów i profesjonalistów o wplywie spolecznej stygmatyzacji na podejmowanie leczenia z powodu zaburzen zwiazanych z hazardem.

OBJECTIVES: Studies carried out so far have shown that negative stereotypes concerning people with gambling disorders and, in particular, the belief that these people bear personal responsibility for their illness, can significantly contribute to the stigmatization of these people. Shame and fear of stigmatization significantly hinder the decision to start treatment. This study investigates the beliefs of respondents about the impact of stigmatization of people with gambling disorders on the social perception of treatment of gambling disorders and the beliefs of respondents about the impact of stigmatization on undertaking treatment or seeking help. Furthermore, the study investigates whether treatment can help reduce stigma and whether professionals, in some way, take this problem into account in their practice. METHODS: In the first half of 2015, 90 semi structured individual interviews were conducted with people with gambling disorders, social workers, therapists employed in addiction treatment facilities, General Practitioners and psychiatrists. RESULTS: The public reaction to the fact of starting treatment depends on how gambling disorder is perceived: in terms of a medical problem or rather in moral terms. Positive reviews were mainly manifested by significant others who, according to Goffman's terminology, are 'wise'. Very often, treatment means having to reveal your problem and face stigmatization. Women in particular are stigmatized because of gambling disorders. This study showed that treatment allows to getr id of guilt by acquiring knowledge on the subject of gambling disorders. CONCLUSIONS: Addressing stigma during early stages of treatment may contribute to the continuity of treatment. Professionals should be aware of their prejudices, as their stigmatizing attitudes can influence treatment outcomes.

The Operative Time for Unilateral Inguinal Hernia Repair in Children Performed with Percutaneous Internal Ring Suturing (PIRS) or Open Approach Method.

In this study, we compared the operative time for unilateral inguinal hernia repair in children performed with either an open approach (OA) or the Percutaneous Internal Ring Suturing (PIRS) method. It was a retrospective chart review of all patients ages 0 to 18 who underwent unilateral inguinal hernia repair in the Department of Pediatric Surgery, Urology and Traumatology of the Regional Hospital in Kielce between January 2011 and December 2018. Patients with bilateral hernias or additional problems were excluded. Of 878 patients qualified for the study, 701 were in the OA group and 177 in the PIRS group. Overall, the time needed to complete the procedure was significantly longer for the OA method. The operative time was longer if the hernia was left-sided (p = 0.024). Analysis by gender showed that operative time was generally longer in males. For both genders, surgery was shorter if the PIRS method was used. For males in the PIRS group the operative time was affected by the location of the hernia, and it was longer for a left-sided hernia. The take-home message is that the PIRS procedure is faster than the OA for inguinal hernia repair in children and it might be considered as a preferred method, especially in females.

Left ventricular diastolic dysfunction and diseases severity contribute to impaired exercise capacity in systemic lupus erythematosus.

OBJECTIVES: Patients with systemic lupus erythematosus (SLE) have a higher risk of myocardial involvement, which can result in ventricular dysfunction. The aim of our study was to estimate potential relationship between exercise capacity assessed by six minute walk test (6MWT) and echocardiographic parameters of left and right ventricular function in SLE patients. METHODS: We prospectively studied 66 SLE patients (57 F, age 44 (20-75) years) and 27 age matched healthy subjects. In addition to routine evaluation, 6MWT and transthoracic echocardiography including LV diastolic dysfunction parameters (E/A, E/É) were performed. RESULTS: While E/A was similar in both groups, E/E' was higher in patients with SLE than in controls, 7.5 (4-22) vs 6.8 (1.6-9.4), p = 0.018. The mean 6MWT distance was significantly shorter in SLE (561.6 ± 150.7 vs 682.6 ± 98.1 m, p < 0.002). Among SLE patients only 53 (80.3%) were capable to walk at least 450 m, while in controls 27 (100%) (p = 0.013). We observed significant correlations between 6MWT distance and SLICC/ACR-DI (rho=-0.44, p < 0.001), E/A (rho = 0.30, p = 0.004), E/E' (rho=-0.36, p < 0.001) in SLE patients. Univariable logistic regression models revealed that SLICC/ACR-DI, E/E', tricuspid regurgitant peak gradient (TRPG), and right ventricular systolic pressure (RVSP) were associated with 6MWT distance lower than < 450 m. ROC curves shown high predictive value of E/E' ratio, TRPG, RVSP in the prediction for 6MWT distance < 450 m. CONCLUSION: Impaired exercise tolerance seems to result mainly from the severity of SLE and LV diastolic dysfunction.

Antimicrobial Properties of Mandelic Acid, Gallic Acid and their Derivatives.

PURPOSE: Alpha-hydroxy acids (AHAs) are one of the classes of hydroxy acids being beneficial for human health. The manuscript summarizes the biological properties of two popular members of AHAs, i.e., Mandelic Acid (MA) and Gallic Acid (GA), with particular emphasis on antimicrobial properties. Moreover, attempts to design new derivatives improving the natural properties of AHAs by using the chemical and physical approach are discussed. METHODS: Antimicrobial properties of MA, an arylalkyl AHA containing phenyl group attached to α- carbon, and GA, an aromatic trihydroxybenzoic acid containing the phenolic ring and carboxylic acid functional group, and their derivatives against common human and plant pathogenic fungi have been reviewed. RESULTS: The antimicrobial activity of MA and GA is a complex phenomenon strictly correlated with other properties exhibited by these acids, e.g., pro-oxidative activity and hydrophobicity. In most cases, the acids derivatives exhibited higher antimicrobial activity than the acids themselves. This is probably because of the higher lipophilicity of moiety that allows better penetration through the cell membrane. CONCLUSION: MA and GA present an excellent health-promoting tool and are valuable starting materials for the design of new compounds such as metal complexes with alkali, or alkali earth metals. The lipophilic, antimicrobial, and pro-oxidative properties act synergistically, supporting the pharmacological and therapeutic effect of acids and their derivatives.

Psychometric properties of the Polish version of two screening tests for gambling disorders: the Problem Gambling Severity Index and Lie/Bet Questionnaire.

To date, no screening tests for gambling disorders have been adapted and validated in Central and Eastern Europe. The aim of this study is to adapt the Problem Gambling Severity Index (PGSI) and Lie/Bet questionnaire (Lie/Bet) and assess their psychometric properties once translated for use with the Polish population. A mixed sample (N = 300) was drawn from venues, social media, snowballing and treatment centers. PGSI had a higher coefficient of predictive power than Lie/Bet. However, differences between validated tests are not significant. Validation of screening tests of gambling disorders showed the necessity for verification of the scale of interpretation of results when conducting tests in Poland, changing cutoff values. The PGSI and Lie/Bet tests are short and easy to apply, they can be implemented in various types of institutions: for screening patients in primary health care facilities and for identifying comorbid gambling disorders in alcohol- and drug-dependence treatment facilities, and in social welfare centers.

HaDeX: an R package and web-server for analysis of data from hydrogen-deuterium exchange mass spectrometry experiments.

MOTIVATION: Hydrogen-deuterium mass spectrometry (HDX-MS) is a rapidly developing technique for monitoring dynamics and interactions of proteins. The development of new devices has to be followed with new software suites addressing emerging standards in data analysis. RESULTS: We propose HaDeX, a novel tool for processing, analysis and visualization of HDX-MS experiments. HaDeX supports a reproducible analytical process, including data exploration, quality control and generation of publication-quality figures. AVAILABILITY AND IMPLEMENTATION: HaDeX is available primarily as a web-server (http://mslab-ibb.pl/shiny/HaDeX/), but its all functionalities are also accessible as the R package (https://CRAN.R-project.org/package=HaDeX) and standalone software (https://sourceforge.net/projects/HaDeX/). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Adequate timing and constant supervision are the keys for successful implementation of levothyroxine or levothyroxine/paracetamol absorption test.

BACKGROUND: Levothyroxine (LT4) pseudomalabsorption due to medication non-adherence results in significant costs for Health Service. High dose LT4 or LT4/paracetamol absorption test is used in such cases. Hence, establishment of an optimal test protocol and timing of sample collection is of utmost importance. CASE PRESENTATION: A 34-year old woman was admitted to our Department because of severe hypothyroidism [on admission thyrotropin (TSH) > 100 µIU/ml, free thyroxine (FT4) 0.13 ng/dl (ref. range 0.93-1.7)] despite apparently taking 1000 µg of LT4 a day. Autoimmune hypothyroidism had been diagnosed 4 years before during post-partum thyroiditis. Subsequently, it was not possible to control her hypothyroidism despite several admissions to two University Hospitals and despite vehement denial of compliance problems. There was no evidence of coeliac disease or other malabsorption problems, though gluten-free and lactose-free diet was empirically instigated without success. A combined paracetamol (1000 mg)/LT4 (1000 µg) absorption test was performed in one of these Hospitals. This showed good paracetamol absorption (from < 2 µg/ml to 14.11 µg/ml at 120 min), with inadequate LT4 absorption (FT4 increase from 5.95 pmol/l to 9.92 pmol/l at 0 and 120 min respectively). About 2 years prior to admission to our Department the patient was treated with escalating doses of levothyroxine [up to 3000 µg of T4 and 40 µg of triiodothyronine (T3) daily] without significant impact on TSH (still > 75 µIU/ml, and FT4 still below reference range).After admission to our Department we performed a 2500 µg LT4 absorption test with controlled ingestion of crushed tablets, strict patient monitoring and sampling at 30 min intervals. We observed a quick and striking increase in FT4 from 0.13 to 0.46, 1.78, 3.05 and 3.81 ng/dl, at 0, 30, 60, 90 and 120 min, respectively. Her TSH concentration decreased to 13.77 µIU/ml within 4 days. When informed, that we had managed to "overcome" her absorption problems, she discharged herself against medical advice and declined psychiatric consultation. CONCLUSIONS: Adequate patient supervision and frequent sampling (e.g. every 30 min for 210 min) is the key for successful implementation of LT4 absorption test. Paracetamol coadministration appears superfluous in such cases.

Synergistic interaction of diclofenac and its metabolites with selected antibiotics and amygdalin in wastewaters.

Diclofenac (DCF), a non-steroidal anti-inflammatory drug (NSAID) belongs to one of the most frequently detected pharmaceutical residues in the environment. Little is known on the interactions of DCF as well as its major biodegradation metabolites 4'-OHDCF and 5-OHDCF with chemical compounds found in wastewater, including antibiotics such as ampicillin and kanamycin. In the present work we examined the potential interactions between DCF, its metabolites 4'-OHDCF and 5-OHDCF and ampicyllin and kanamycin. We also measured the effect of the mixture of DCF with natural compound - amygdalin. We evaluated the following parameters: E. coli K-12 cells viability, growth inhibition of E. coli K-12 culture, genotoxicity, oxidative stress parameters: sodA promoter induction and ROS generation. The reactivity of E. coli SM recA:luxCDABE biosensor strain in wastewaters matrices contaminated with DCF and kanamycin was also monitored. Obtained results indicated that used antibiotics (ampicyllin, kanamycin) enhanced the toxic effect of DCF used individually and in the mixtures with its metabolites 4'-OHDCF and 5-OHDCF toward E. coli. Similar effect was also obtained in genotoxicity assay. The oxidative stress assays revealed that the highest level of ROS generation and sodA promoter induction were obtained also for the mixtures of DCF, its metabolites with antibiotics. It was also showed that amygdalin influenced the activity of DCF and its biodegradation metabolites. The strongest luminescence response of E. coli SM biosensor strain with recA:luxCDABE genetic construct in filtered treated wastewaters, comparable to control sample was noticed. Obtained results showed that DCF and its biodegradation metabolites 4'-OHDCF and 5-OHDCF can interact with tested antibiotics and compounds of natural origin, i.e. amygdalin to form mixtures showing stronger antimicrobial activity against E. coli than parent chemicals. Moreover the assays in wastewater matrices revealed that E. coli SM recA:luxCDABE biosensor strains is a good tool for bacteria monitoring in wastewater environments.

Calcium ions modulate the structure of the intrinsically disordered Nucleobindin-2 protein.

Nucleobindin-2 (Nucb2) is a widely expressed multi-domain protein. Nucb2 participates in many physiological processes, i.e. calcium level maintenance, feeding regulation in the hypothalamus, emotion and stress regulation, and many others. To date, this protein has not been structurally characterized. We describe the first comparative structural analysis of two homologs, a Gallus gallus and a Homo sapiens Nucb2. The in silico analysis suggested that apo-Nucb2s contain a mosaic-like structure, consisting of intertwined disordered and ordered regions. Surprisingly, the hydrogen-deuterium exchange mass spectrometry results revealed that Nucb2 is divided into two parts: an N-terminal half with a stable mosaic-like structure and a disordered C-terminal half. However, the presence of Ca2+ induces the formation of a mosaic-like structure in the C-terminal half of the Nucb2s. The Ca2+ also affects the tertiary and quaternary structure of Nucb2s. The presence of Ca2+ leads to an overall compaction of the Nucb2 molecule, resulting in structural change that is propagated along the molecule, which in turn affects the quaternary structure of the protein. Intrinsic disorder, and the mosaic-like Ca2+ dependent structure of Nucb2s, might be seen as the molecular factors responsible for their multifunctionality. Thus, Nucb2s might function as the versatile Ca2+ sensor involved in signal transduction.

The study of biological activity of transformation products of diclofenac and its interaction with chlorogenic acid.

In the present work we compared the biological activity of DCF, 4'-OHDCF and 5-OHDCF as molecules of most biodegradation pathways of DCF and selected transformation products (2-hydroxyphenylacetic acid; 2,5-dihydroxyphenylacetic acid and 2,6-dichloroaniline) which are produced during AOPs, such as ozonation and UV/H2O2. We also examined the interaction of DCF with chlorogenic acid (CGA). CGA is commonly used in human diet and entering the environment along with waste mainly from the processing and brewing of coffee and it can be toxic for microorganisms included in activated sludge. In the present experiment the evaluation of following parameters was performed: E. coli K-12 cells viability, growth inhibition of E. coli K-12 culture, LC50 and mortality of Chironomus aprilinus, genotoxicity, sodA promoter induction and ROS generation. In addition the reactivity of E. coli SM recA:luxCDABE biosensor strain in wastewater matrices was measured. The results showed the influence of DCF, 4'-OHDCF and 5-OHDCF on E. coli K-12 cells viability and bacteria growth, comparable to AOPs by-products. The highest toxicity was observed for selected, tested AOPs by-products, in comparison to the DCF, 4'-OHDCF and 5-OHDCF. Genotoxicity assay indicated that 2,6-dichloroaniline (AOPs by-product) had the highest toxic effect. The oxidative stress assays revealed that the highest level of ROS generation and sodA promoter induction were obtained for DCF, 4'-OHDCF and 5-OHDCF, compared to other tested compounds. We have also found that there is an interaction between chlorogenic acid and DCF, which resulted in increased toxicity of the mixture of the both compounds to E. coli K-12, comparable to parent chemicals. The strongest response of E. coli SM biosensor strain with recA:luxCDABE genetic construct in filtered treated wastewaters, comparable to control sample was noticed. It indicates, that E. coli SM recA:luxCDABE biosensor strains is a good tool for bacteria monitoring in wastewater environment. Due to toxicity and biological activity of tested DCF transformation products, there is a need to use additional wastewater treatment systems for wastewater contaminated with pharmaceutical residues.