Polyamines are essential compounds to all living organisms and in the specific case of Trypanosoma cruzi, the causative agent of Chagas disease, they are exclusively obtained through transport ...processes since this parasite is auxotrophic for polyamines. Previous works reported that retinol acetate inhibits Leishmania growth and decreases its intracellular polyamine concentration. The present work describes a combined strategy of drug repositioning by virtual screening followed by in vitro assays to find drugs able to inhibit TcPAT12, the only polyamine transporter described in T. cruzi. After a screening of 3000 FDA-approved drugs, 7 retinoids with medical use were retrieved and used for molecular docking assays with TcPAT12. From the docked molecules, isotretinoin, a well-known drug used for acne treatment, showed the best interaction score with TcPAT12 and was selected for further in vitro studies. Isotretinoin inhibited the polyamine transport, as well as other amino acid transporters from the same protein family (TcAAAP), with calculated IC50 values in the range of 4.6-10.3 μM. It also showed a strong inhibition of trypomastigote burst from infected cells, with calculated IC50 of 130 nM (SI = 920) being significantly less effective on the epimastigote stage (IC50 = 30.6 μM). The effect of isotretinoin on the parasites plasma membrane permeability and on mammalian cell viability was tested, and no change was observed. Autophagosomes and apoptotic bodies were detected as part of the mechanisms of isotretinoin-induced death indicating that the inhibition of transporters by isotretinoin causes nutrient starvation that triggers autophagic and apoptotic processes. In conclusion, isotretinoin is a promising trypanocidal drug since it is a multi-target inhibitor of essential metabolites transporters, in addition to being an FDA-approved drug largely used in humans, which could reduce significantly the requirements for its possible application in the treatment of Chagas disease.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Crystal violet (CV) was used for several years in blood banks to eliminate the parasite Trypanosoma cruzi in endemic areas in order to prevent transfusion-transmitted Chagas disease. One mechanism of ...action described for CV involves inhibition of proline uptake. In T. cruzi, proline is essential for host cell infection and intracellular differentiation among other processes, and can be obtained through the proline permease TcAAAP069.
CV inhibited proline transporter TcAAAP069 and parasites overexpressing this permease were 47-fold more sensitive to this compound than control parasites. Using CV as reference molecule, loratadine, cyproheptadine, olanzapine and clofazimine were identified as structurally related compounds to CV (structural analogues) by in silico drug repurposing through a similarity-based virtual screening protocol. All these already-approved drugs for clinical use inhibited TcAAAP069 activity with different efficacies and also presented trypanocidal action in epimastigotes, trypomastigotes and amastigotes of the Y, CL Brener and Dm28c T. cruzi strains. Finally, a synergistic effect between benznidazole and the CV chemical analogues was evidenced by combination and dose-reduction indexes values in epimastigotes and trypomastigotes of the Y strain.
Loratadine, cyproheptadine and clofazimine inhibit TcAAAP069 proline transporter and also present trypanocidal effect against all T. cruzi life stages in strains from three different DTUs. These CV structural analogues could be a starting point to design therapeutic alternatives to treat Chagas disease by finding new indications for old drugs. This approach, called drug repurposing is a recommended strategy by the World Health Organization to treat neglected diseases, like Chagas disease, and combination therapy may improve the possibility of success of repositioned drugs.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Trypanosoma cruzi, the etiological agent of Chagas' disease, has a metabolism largely based on the consumption of glucose and proline. This amino acid is essential for host cells infection and ...intracellular differentiation. In this work we identified a proline transporter (TcAAAP069) by yeasts complementation assays and overexpression in Trypanosoma cruzi epimastigotes. TcAAAP069 is mono-specific for proline but presents an unusual feature; the lack of stereospecificity, because it is competitively inhibited by the D- enantiomer. Parasites overexpressing TcAAAP069 have an increased intracellular proline concentration, 2.6-fold higher than controls, as a consequence of a higher proline transport rate. Furthermore, augmented proline concentration correlates with an improved resistance to trypanocidal drugs and also to reactive oxygen species including hydrogen peroxide and nitric oxide, emulating natural physiological situations. The IC50s for nifurtimox, benznidazole, H2O2 and NO. were 125%, 68%, 44% and 112% higher than controls, respectively. Finally, proline metabolism generates a higher concentration (48%) of ATP in TcAAAP069 parasites. Since proline participates on essential energy pathways, stress and drug resistance responses, these results provide a novel target for the development of new drugs for the treatments for Chagas' disease.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Seawater desalination by reverse osmosis is growing exponentially due to water scarcity. Byproducts of this process (e.g. brines), are generally discharged directly into the coastal ecosystem, ...causing detrimental effects, on benthic organisms. Understanding the cellular stress response of these organisms (biomarkers), could be crucial for establishing appropriate salinity thresholds for discharged brines. Early stress biomarkers can serve as valuable tools for monitoring the health status of brine-impacted organisms, enabling the prediction of long-term irreversible damage caused by the desalination industry. In this study, we conducted laboratory-controlled experiments to assess cellular and molecular biomarkers against brine exposure in two salinity-sensitive Mediterranean seagrasses: Posidonia oceanica and Cymodocea nodosa. Treatments involved exposure to 39, 41, and 43 psu, for 6 h and 7 days. Results indicated that photosynthetic performance remained unaffected across all treatments. However, under 43 psu, P. oceanica and C. nodosa exhibited lipid oxidative damage, which occurred earlier in P. oceanica. Additionally, P. oceanica displayed an antioxidant response at higher salinities by accumulating phenolic compounds within 6 h and ascorbate within 7 d; whereas for C. nodosa the predominant antioxidant mechanisms were phenolic compounds accumulation and total radical scavenging activity, which was evident after 7 d of brines exposure. Finally, transcriptomic analyses in P. oceanica exposed to 43 psu for 7 days revealed a poor up-regulation of genes associated with brassinosteroid response and abiotic stress response, while a high down-regulation of genes related to primary metabolism was detected. In C. nodosa, up-regulated genes were involved in DNA repair, cell cycle regulation, and reproduction, while down-regulated genes were mainly associated with photosynthesis and ribosome assembly. Overall, these findings suggest that 43 psu is a critical salinity-damage threshold for both seagrasses; and despite the moderate overexpression of several transcripts that could confer salt tolerance, genes involved in essential biological processes were severely downregulated.
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•P. oceanica and C. nodosa suffer oxidative damage at 43 psu.•Both seagrasses display different antioxidant mechanisms when exposed to brine.•RNAseq in P. oceanica reveal an up-regulation of brassinosteroid response genes.•RNAseq in C. nodosa reveal an up-regulation of DNA repair and cell cycle genes.•Genes involved in essential biological processes are severely downregulated.
Background and purpose
The COVID‐19 (SARS‐CoV‐2) outbreak has disrupted residency programmes due to university and hospitals’ priorities to face this emergency at all cost. Most research projects and ...clinical trials were temporarily stopped or postponed. The Resident and Research Fellow Section (RRFS) of the European Academy of Neurology (EAN) has decided to assess the impact of the COVID‐19 pandemic on neurology training.
Methods
All EAN RRFS members were invited to fill out an online questionnaire of 40 items concerning their clinical involvement during the COVID‐19 emergency, and the impact of the pandemic on their training (Appendix S1).
Results
Of the 227 RRFS members who completed the questionnaire, 222 were from Europe, and of those 111 were from Portugal, Italy or France. Responders highlighted that severe restrictions have been imposed to face this pandemic, including reduction of inpatient beds, prohibition of in‐person visits and limitation to hospital access for patients’ relatives. This was accompanied by an increase in email correspondence and phone calls with 50% of countries allowing telemedicine to reach outpatients. Seventy‐nine per cent of the respondents felt that the pandemic will probably have a serious impact on their training and career.
Conclusion
The pandemic led to a disruption of neurology activities, including medical training and research. The long‐run impact of these changes remains unknown, but it will probably change the way neurology practice and training will be organized for future generations.
The first impact of the COVID‐19 pandemic. Data from residents and research fellows of 30 European countries on the modifications of their residency programmes and research plans.
•Isotretinoin, drug used for severe acne, is trypanocidal in vitro at nanomolar concentrations.•Isotretinoin administration in the chronic phase of T. cruzi infected mice abolished ...parasitemia.•Isotretinoin improved myocardial compromise in mice.
Benznidazole and nifurtimox are the drugs currently used for the treatment of Chagas disease, however its side effects may affect patient adherence. In the search for new alternative therapies, we previously identified isotretinoin (ISO), an FDA-approved drug widely used for the treatment of severe acne through a drug repurposing strategy. ISO shows a strong activity against Trypanosoma cruzi parasites in the nanomolar range, and its mechanism of action is through the inhibition of T. cruzi polyamine and amino acid transporters from the Amino Acid/Auxin Permeases (AAAP) family. In this work, a murine model of chronic Chagas disease (C57BL/6 J mice), intraperitoneally infected with T. cruzi Nicaragua isolate (DTU TcI), were treated with different oral administrations of ISO: daily doses of 5 mg/kg/day for 30 days and weekly doses of 10 mg/kg during 13 weeks. The efficacy of the treatments was evaluated by monitoring blood parasitemia by qPCR, anti-T. cruzi antibodies by ELISA, and cardiac abnormalities by electrocardiography. No parasites were detected in blood after any of the ISO treatments. The electrocardiographic study of the untreated chronic mice showed a significant decrease in heart rate, while in the treated mice this negative chronotropic effect was not observed. Atrioventricular nodal conduction time in untreated mice was significantly longer than in treated animals. Mice treated even with ISO 10 mg/kg dose every 7 days, showed a significant reduction in anti-T. cruzi IgG levels. In conclusion, the intermittent administration of ISO 10 mg/kg would improve myocardial compromise during the chronic stage.
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Hepatitis C chronic liver disease is a major cause of liver transplant in developed countries. This article reports the first nationwide population-based survey conducted to estimate the ...seroprevalence of HCV antibodies and associated risk factors in the urban population of Brazil.
The cross sectional study was conducted in all Brazilian macro-regions from 2005 to 2009, as a stratified multistage cluster sample of 19,503 inhabitants aged between 10 and 69 years, representing individuals living in all 26 State capitals and the Federal District. Hepatitis C antibodies were detected by a third-generation enzyme immunoassay. Seropositive individuals were retested by Polymerase Chain Reaction and genotyped. Adjusted prevalence was estimated by macro-regions. Potential risk factors associated with HCV infection were assessed by calculating the crude and adjusted odds ratios, 95% confidence intervals (95% CI) and p values. Population attributable risk was estimated for multiple factors using a case-control approach.
The overall weighted prevalence of hepatitis C antibodies was 1.38% (95% CI: 1.12%-1.64%). Prevalence of infection increased in older groups but was similar for both sexes. The multivariate model showed the following to be predictors of HCV infection: age, injected drug use (OR = 6.65), sniffed drug use (OR = 2.59), hospitalization (OR = 1.90), groups socially deprived by the lack of sewage disposal (OR = 2.53), and injection with glass syringe (OR = 1.52, with a borderline p value). The genotypes 1 (subtypes 1a, 1b), 2b and 3a were identified. The estimated population attributable risk for the ensemble of risk factors was 40%. Approximately 1.3 million individuals would be expected to be anti-HCV-positive in the country.
The large estimated absolute numbers of infected individuals reveals the burden of the disease in the near future, giving rise to costs for the health care system and society at large. The known risk factors explain less than 50% of the infected cases, limiting the prevention strategies. Our findings regarding risk behaviors associated with HCV infection showed that there is still room for improving strategies for reducing transmission among drug users and nosocomial infection, as well as a need for specific prevention and control strategies targeting individuals living in poverty.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
In nature 2‐deoxy‐D‐ribose‐5‐phosphate aldolase (DERA) catalyses the reversible formation of 2‐deoxyribose 5‐phosphate from D‐glyceraldehyde 3‐phosphate and acetaldehyde. In addition, this enzyme can ...use acetaldehyde as the sole substrate, resulting in a tandem aldol reaction, yielding 2,4,6‐trideoxy‐D‐erythro‐hexapyranose, which spontaneously cyclizes. This reaction is very useful for the synthesis of the side chain of statin‐type drugs used to decrease cholesterol levels in blood. One of the main challenges in the use of DERA in industrial processes, where high substrate loads are needed to achieve the desired productivity, is its inactivation by high acetaldehyde concentration. In this work, the utility of different variants of Pectobacterium atrosepticum DERA (PaDERA) as whole cell biocatalysts to synthesize 2‐deoxyribose 5‐phosphate and 2,4,6‐trideoxy‐D‐erythro‐hexapyranose was analysed. Under optimized conditions, E. coli BL21 (PaDERA C‐His AA C49M) whole cells yields 99 % of both products. Furthermore, this enzyme is able to tolerate 500 mM acetaldehyde in a whole‐cell experiment which makes it suitable for industrial applications.
Using whole cells of E. coli BL21 (PaDERA C‐His AA C49M) as biocatalyst, a 99% yield of both 2‐deoxyribose‐5‐phosphate and 2,4,6‐trideoxy‐D‐erythro‐hexapyranose was obtained. Furthermore, this enzyme is capable of tolerating up to 500 mM acetaldehyde in a whole‐cell experiment, making it suitable for industrial applications.
Polyamines play critical roles as regulators of cell growth and differentiation. In contrast with other protozoa, the human parasite Trypanosoma cruzi, the etiological agent of Chagas disease, is ...auxotrophic for polyamines. Therefore, their intracellular availability depends exclusively on polyamine transport and inhibition of these uptake processes can alter the viability of the parasite. The polyamine analogues used in this work were successfully tested as antiproliferative agents in cancer cells, bacteria, fungi and also showed a potent antiplasmodial effect. We evaluated the activity of these compounds on polyamine transport in T. cruzi and assessed the effects on parasite viability. Three polyamine derivatives, AMXT1501, Ant4 and Ant44, inhibited the putrescine transport in epimastigotes (the insect stage of T. cruzi) with calculated IC50 values of 2.43, 5.02 and 3.98 μM, respectively. In addition, only Ant4 and Ant44 inhibited spermidine transport with IC50 of 8.78 μM and 13.34 μM, respectively. The Ant4 analogue showed a high trypanocidal effect on trypomastigotes (the bloodstream stage of T. cruzi) with an IC50 of 460 nM, (SI = 12.7) while in epimastigotes the IC50 was significantly higher (16.97 μM). In addition, we studied the effect of the combination of benznidazole, a drug used in treating Chagas disease, with Ant4 on the viability of epimastigotes. The combined treatment produced a significant increase on the inhibition of parasites growth compared with individual treatments. In summary, these results suggest that Ant4, a putrescine conjugate, is a promising compound for the treatment of Chagas disease because it showed a potent trypanocidal effect via its inhibition of polyamine import.
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•Trypanosoma cruzi is auxotrophic for polyamines (PA) and depends on their transport.•Eight PA analogs were evaluated as PA transport inhibitors.•The PA analogs AMXT 1501, Ant4 and Ant44 inhibited PA transport.•Ant4 also showed a high trypanocidal effect alone or combined with benznidazole.•Ant4 is a promising compound for the treatment of Chagas disease.
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