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The prion protein (PrP) is implicitly involved in the pathogenesis of transmissible spongiform encephalopathies (TSEs). The conversion of normal cellular PrP (PrP C), a protein that is predominantly α-helical, to a β-sheet-rich isoform (PrP Sc), which has a propensity to aggregate, is the key molecular event in prion diseases. During its short life span, PrP can experience two different pH environments; a mildly acidic environment, whilst cycling within the cell, and a neutral pH when it is glycosyl phosphatidylinositol (GPI)-anchored to the cell membrane. Ion mobility (IM) combined with mass spectrometry has been employed to differentiate between two conformational isoforms of recombinant Syrian hamster prion protein (SHaPrP). The recombinant proteins studied were α-helical SHaPrP(90-231) and β-sheet-rich SHaPrP(90-231) at pH 5.5 and pH 7.0. The recombinant proteins have the same nominal mass-to-charge ratio ( m/z) but differ in their secondary and tertiary structures. A comparison of traveling-wave (T-Wave) ion mobility and drift cell ion mobility (DCIM) mass spectrometry estimated and absolute cross-sections showed an excellent agreement between the two techniques. The use of T-Wave ion mobility as a shape-selective separation technique enabled differentiation between the estimated cross-sections and arrival time distributions (ATDs) of α-helical SHaPrP(90-231) and β-sheet-rich SHaPrP(90-231) at pH 5.5. No differences in cross-section or ATD profiles were observed between the protein isoforms at pH 7.0. The findings have potential implications for a new ante-mortem screening assay, in bodily fluids, for prion misfolding diseases such as TSEs. Two different prion protein (PrP) conformational isoforms, representative of healthy and diseased PrP, were analyzed by means of ion mobility mass spectrometry.

Cancer and infectious diseases are major causes of global morbidity and mortality stressing the need to find novel drugs with promising dual anticancer and antimicrobial efficacy. Gold complexes have been studied for the past years due to their anticancer properties, with a few of them displaying antimicrobial properties, which support their pharmacological interest. Within this scope, we investigated six gold bisdithiolate complexes [Au (bdt)2]− (1), [Au (dcbdt)2]− (2), [Au (3-cbdt)2]− (3), [Au (4-cbdt)2]− (4), [Au (pdt)2]− (5) and [Au (dcdmp)2]− (6), and) against the ovarian cancer cell lines A2780 and A2780cisR, the Gram-positive bacteria Staphylococcus aureus Newman, the Gram-negative bacteria Escherichia coli ATCC25922 and Burkholderia contaminans IST408, and the pathogenic yeasts Candida glabrata CBS138 and Candida albicans SC5134. Complexes 2 and 6, with ligands containing aromatic pyrazine or phenyl rings, substituted with two cyanonitrile groups, showed after 24 h of incubation high anticancer activities against A2780 ovarian cancer cells (IC50~5 µM), being also able to overcome cisplatin resistance in A2780cisR cells. Both complexes induced the formation of ROS, activated caspase-3/7, and induced necrosis (LDH release) in a dose-dependent way, in a greater extent in the case of 6. Among the bacterial and fungal strains tested, only complex 6 presented antimicrobial activity against S. aureus Newman, indicating that this complex is a potential novel anticancer and antibacterial agent. These results delve into the structure-activity relationship of the complexes, considering molecular alterations such as replacing a phenyl group for a pyrazine group, and the inclusion of one or two cyanonitrile appendage groups, and their effects on biological activity. Overall, both complexes were found to be promising leads for the development of future anticancer drugs against low sensitive or cisplatin resistant tumors.

Quando a 25 de abril de 1974 representantes do Movimento das Forças Armadas (MFA) entraram no quartel do Carmo em Lisboa para receber a capitulação de Marcelo Caetano, caía em um só dia a mais longa ditadura do século xx da Europa Ocidental. Perante uma opinião pública internacional apreensiva, o programa do MFA punha fim de forma inequívoca a 48 anos de ditadura e abria caminho para o processo de descolonização e democrati-zação do país. No entanto, o que se seguiu à revolução esteve longe de ser um processo linear de democratização. Se a queda da ditadura se tornou rapidamente consensual entre vastas faixas da população e do espectro político, as divergências sobre o caminho político a seguir dividiram o país durante dois anos, marcados por instabilidade política e social, com fortes oposições partidárias, manifestações, nacionalizações, ocupações de terras e fábricas e dois golpes de Estado. Uma das conclusões que a perspetiva histórica e comparada permite é a de que, apesar do ambiente crispado que se viveu entre abril de 1974 e abril de 1976, o período revolucionário foi decisivo na reconstrução política do país, ao consolidar as bases ideológicas da democracia em Portugal, assentes na condenação de regimes autoritários e na aposta pelo pluripartidarismo das democracias ocidentais.

Background and objectives: Polyvascular atherosclerosis is frequent and associated with a high cardiovascular risk, although the mechanisms regulating the atherosclerosis extent to single or multiple arterial territories are still poorly understood. Inflammation regulates atherogenesis and soluble CD40 ligand (sCD40L) is an inflammatory mediator associated with the presence of single-territorial atherosclerosis. We assessed whether the sCD40L expression is associated with the atherosclerosis extent to single or multiple arterial territories and with the atherosclerosis severity in different territories. Materials and Methods: We prospectively enrolled 94 participants with no atherosclerosis (controls, n = 26); isolated coronary atherosclerosis (group 1, n = 20); coronary and lower extremity (LE) atherosclerosis (group 2, n = 18); coronary and carotid atherosclerosis (group 3, n = 12); and coronary, LE, and carotid atherosclerosis (group 4, n = 18). Serum sCD40L levels were quantified. Results: The sCD40L levels (ng/mL, mean (standard deviation)) were 4.0 (1.5), 5.6 (2.6), 7.2 (4.2), 5.9 (3.7), and 5.1 (2.4) in controls and groups 1 to 4, respectively (ANOVA p = 0.012). In nonrevascularized patients, the sCD40L levels were significantly higher in group 2 than in group 1 and were correlated with the number of LE diseased segments. Prior LE bypass surgery was associated with lower sCD40L levels. Coexistence of coronary and LE atherosclerosis was independently associated with the sCD40L levels. Conclusions: The sCD40L levels were increased in stable atherosclerosis, particularly in polyvascular coronary and LE atherosclerosis. The number of LE diseased segments and prior LE revascularization were associated with sCD40L expression. To our knowledge, these are novel data, which provide insights into the mechanisms underlying multi-territorial atherosclerosis expression. sCD40L may be a promising noninvasive tool for refining the stratification of the systemic atherosclerotic burden.

Surface properties of composites such as roughness and color impact periodontal health and aesthetic outcomes. Novel bulk-fill composites with improved functionality are being introduced and, in light of the existing variety of finishing/polishing procedures, research of their surface properties is warranted. Sixty discs were prepared from bulk-fill composites (Filtek™ Bulk Fill Posterior Restorative and Fill-Up™) and incremental-fill Filtek™ Z250. They were further divided according to different polishing procedures (n = 5): three multi-step polishing procedures or finishing with a bur (control). Surface roughness (Ra) was measured using an atomic force microscope (The AFM Workshop TT-AFM). A spectrophotometer (Spectroshade Micro Optic) was used to determine color stability, after exposure to a coffee solution. Data were analyzed using two-way MANOVA (significance level of 5%). Resin composite type, polishing procedure, and their interaction had a statistically significant effect on surface roughness (p < 0.001) and color change (p < 0.001). Fill-Up™ exhibited the highest surface roughness and greatest color change. Differences in color change were statistically significant (p < 0.001). Filtek™ Bulk Fill registered the lowest surface roughness and color change, after the three-step polishing procedure. Both parameters were significantly correlated (ρ = 0.754, p < 0.001) and found to be material dependent and polishing-procedure dependent. Higher surface roughness relates to greater color changes.

The Joanes I Reservoir is responsible for 40% of the drinking water supply of the Metropolitan Region of Salvador, Bahia, Brazil. For water sources such as this, there is concern regarding the proliferation of potentially toxin-producing cyanobacteria, which can cause environmental and public health impacts. To evaluate the presence of cyanobacteria and their cyanotoxins in the water of this reservoir, the cyanobacteria were identified by microscopy; the presence of the genes of the cyanotoxin-producing cyanobacteria was detected by molecular methods (polymerase chain reaction (PCR)/sequencing); and the presence of toxins was determined by liquid chromatography with tandem mass spectrometry (LC-MS/MS). The water samples were collected at four sampling points in the Joanes I Reservoir in a monitoring campaign conducted during the occurrence of phytoplankton blooms, and the water quality parameters were also analysed. Ten cyanobacteria species/genera were identified at the monitoring sites, including five potentially cyanotoxin-producing species, such as Cylindrospermopsis raciborskii, Cylindrospermopsis cf. acuminato-crispa, Aphanocapsa sp., Phormidium sp., and Pseudanabaena sp. A positive result for the presence of the cylindrospermopsin toxin was confirmed at two sampling points by LC-MS/MS, which indicated that the populations are actively producing toxins. The analysis of the PCR products using the HEPF/HEPR primer pair for the detection of the microcystin biosynthesis gene mcyE was positive for the analysed samples. The results of this study point to the worrisome condition of this reservoir, from which water is collected for public supply, and indicate the importance of the joint use of different methods for the analysis of cyanobacteria and their toxins in reservoir monitoring.

Background: Ferrabis(dicarbollide) ([o-FESAN]−) in combination with proton–boron fusion therapy (PBFT) or boron neutron capture therapy (BNCT) are promising alternative radiation modalities for the treatment of breast cancer. The aim of this study was to explore the underlying effects of [o-FESAN]− radio enhancement on breast cancer cells in vitro and in vivo, and to perform comparative dosimetry calculations. Methods: The cellular effects on SKBR-3 and MDA-MB-231 breast cancer cells and MDA-MB-231 xenograft-bearing nude mice induced by carrier-free [o-FESAN]− after BNCT or PBFT were evaluated following recommended protocols. Monte Carlo (MC) dosimetry calculations were performed at the cellular scale for both radiation modalities. Results: Selective retention of [o-FESAN]− within the cytoplasm and nucleus of SKBR-3 and MDA-MB-231 breast cancer cells is demonstrated. Moreover, in vivo studies with MDA-MB-231 xenograft-bearing nude mice show appreciable accumulation of [o-FESAN]− in the tumor. Both radiation modalities induce loss of cellular viability and survival. Comparative dosimetry studies between proton and neutron irradiation agree with the viability data, showing a good correlation between absorbed dose vs. cellular effects. In the case of PBFT, cell structural changes are likely due to necrosis caused by the production of reactive oxygen species (ROS). To explain the radio enhancement effects in more detail, other mechanisms should be taken into consideration. Conclusions: Our results validate the effectiveness of both PBFT and BNCT therapeutic modalities, warranting further studies on carrier-free [o-FESAN]− as a candidate drug for potential clinical translation of radio enhancers in binary radiation therapies.

Transmissible spongiform encephalopathies are characterised by widespread deposition of fibrillar and/or plaque-like forms of the prion protein. These aggregated forms are produced by misfolding of the normal prion protein, PrP(C), to the disease-associated form, PrP(Sc), through mechanisms that remain elusive but which require either direct or indirect interaction between PrP(C) and PrP(Sc) isoforms. A wealth of evidence implicates other non-PrP molecules as active participants in the misfolding process, to catalyse and direct the conformational conversion of PrP(C) or to provide a scaffold ensuring correct alignment of PrP(C) and PrP(Sc) during conversion. Such molecules may be specific to different scrapie strains to facilitate differential prion protein misfolding. Since molecular cofactors may become integrated into the growing protein fibril during prion conversion, we have investigated the proteins contained in prion disease-specific deposits by shotgun proteomics of scrapie-associated fibrils (SAF) from mice infected with 3 different strains of mouse-passaged scrapie. Concomitant use of negative control preparations allowed us to identify and discount proteins that are enriched non-specifically by the SAF isolation protocol. We found several proteins that co-purified specifically with SAF from infected brains but none of these were reproducibly and demonstrably specific for particular scrapie strains. The α-chain of Na(+)/K(+)-ATPase was common to SAF from all 3 strains and we tested the ability of this protein to modulate in vitro misfolding of recombinant PrP. Na(+)/K(+)-ATPase enhanced the efficiency of disease-specific conversion of recombinant PrP suggesting that it may act as a molecular cofactor. Consistent with previous results, the same protein inhibited fibrillisation kinetics of recombinant PrP. Since functional interactions between PrP(C) and Na(+)/K(+)-ATPase have previously been reported in astrocytes, our data highlight this molecule as a key link between PrP function, dysfunction and misfolding.

Background Protons, which are considered low-LET (Linear Energy Transfer) radiation, have an average RBE (relative biological effectiveness) of 1.1, with a range from 0.7 to 1.6. Thus, increasing biological effectiveness is of high interest in radiation oncology, and one way to enhance this is by using radiosensitizers. The present work investigates the effectiveness of the proton boron fusion reaction (PBFR) at the cellular level, using the sodium salt of metallacarborane [3,3’-Co(C2B9H11)2] − (Na[o-COSAN]) as the boron source, aiming to explore the potential of this type of boron clusters as a radiosensitizer for proton therapy. Therefore, the main goal was to test the hypothesis that loading the cells with boron will favour the PBFR at energies close to the Bragg peak. This would enhance the radiation-induced biological effects through the production of alpha-particles. Results MDA-MB-231 breast cancer cells were used. Nuclear microscopy assessed [o-COSAN] uptake and distribution in single cells, while biodistribution was studied in tumor-bearing Balb/cSlc-nu/nu mice (MDA-MB-231 xenograft), with boron accumulation in target organs and tumor measured by ICP-OES. The cells were irradiated with a proton beam tuned to reach the PBFR resonance energy of 675 keV at the cell layer. DNA damage was assessed with the g-H2AX assay and cell survival with the clonogenic assay. Beam parameters and dose calibration curves using radiochromic films validated Monte Carlo dosimetry simulations. As expected, we observed higher biological damage in irradiated cells and the presence of [o-COSAN] − potentiated the damage. These results translate into a lower cellular viability, indicating that DNA damage imposed colonies smaller than their non-irradiated counterparts. This suggests that these damages either took longer time to be repaired or made the cells undergo less efficient survival mechanisms. Conclusions The radiosensitizing effect of [o-COSAN] − by strategic cellular 11 B placement and proton irradiation intensifies the DNA damage, making the nucleus particularly susceptible and thus increasing the destructive capability of alpha-particles, generated in the nuclear fusion reaction, which may lead to increased cell mortality.

Background: Radiosensitizers can be used to enhance tumor response and mitigate toxicity in healthy tissues during radiation therapy. This study investigates the radiosensitizing potential of the metallacarborane Fe/57Fe-ferrabisdicarbollide in SK-BR-3 and MDA-MB-231 breast cancer cells, using two distinct gamma-photon sources: high-dose 60Co (2.08 Gy) and low-dose 57Co (37.55 mGy, 57Fe Mössbauer effect). Methods: We evaluated cell viability and survival in 2D monolayer and 3D spheroid cultures, as well as the mechanism of cell death (ROS production, apoptosis or necrosis). Computational dosimetry was used to calculate the average absorbed dose. Results: In 2D models, both radiation sources induced reduced viability and increased ROS, with distinct cell death patterns dependent on the source (apoptosis or necrosis). Comparing 2D and 3D MDA-MB-231 models revealed that spheroid survival was significantly more impaired. The low-dose 57Co source caused a significant radiosensitization in MDA-MB-231 spheroids, dramatically impacting viability and survival. This effect is attributed to the Mössbauer effect, where the resonant absorption of 14.41 keV radiation by 57Fe leads to a massive, localized dose enhancement. The subsequent cascade of Auger and conversion electrons (local high LET) caused significantly greater cellular damage than sparse photon radiation. Conclusions: Fe/57Fe-ferrabisdicarbollide demonstrates a potent radiosensitizing effect depending on the cell model and the radiation source used. Crucially, the observed radiosensitization allows for the development of a new, more efficient cancer radiotherapy approach that can achieve therapeutic efficacy using a significantly lower radiation dose to the patient. This paves the way for safer and better-tolerated cancer treatments.