Measuring Protein-Protein Interactions and Quantifying Their Dissociation Constants with Mass Photometry.

Protein-protein interactions underlie most biological processes, and determining the affinity and abundance of binding partners for each interaction is often a challenging task because these interactions often involve multiple ligands and binding sites. Standard methods for determining the affinity of protein interactions often require a large amount of starting material in addition to potentially disruptive labeling or immobilization of the binding partners. Mass photometry is a bioanalytical technique that measures the mass of single biomolecules in solution, quickly and with minimal sample requirements. This article describes how mass photometry can be used to determine the mass distribution of binding partners, the complexes they form, the relative abundance of each species, and, accordingly, the dissociation constant (KD ) of their interactions. © 2024 Refeyn Ltd. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Using mass photometry to measure protein-protein binding and quantify the KD of this interaction.

New Perspectives on the Sustainable Employment of Chestnut Shells as Active Ingredient against Oral Mucositis: A First Screening.

Oral mucositis (OM), a common side effect of oncological treatment, is an oral mucosal disorder characterized by painful ulcerations and increased risk of infection. The use of natural antioxidants to suppress the redox imbalance responsible for the OM condition has emerged as an interesting approach to prevent/treat OM. This study aims to explore the chestnut (Castana sativa) shells as potential active ingredient against OM. Therefore, chestnut shells were extracted at different temperatures (110-180 °C) by Subcritical Water Extraction (SWE), aiming to recover antioxidants. The extracts were also evaluated against microorganisms present in the oral cavity as well as on human oral cell lines (TR146 and HSC3). The highest phenolic content was obtained with the extraction temperature of 110 °C, exhibiting the best antioxidant/antiradical activities and scavenging efficiencies against HOCl (IC50 = 4.47 µg/mL) and ROO• (0.73 µmol TE/mg DW). High concentrations of phenolic acids (e.g., gallic and protocatechuic acids) and flavanoids (catechin, epicatechin and rutin) characterized the phenolic profile. The antimicrobial activity against several oral microorganisms present in the oral cavity during OM, such as Streptococcus, Staphylococcus, Enterococcus, and Escherichia, was demonstrated. Finally, the effects on HSC3 and TR146 cell lines revealed that the extract prepared at 110 °C had the lowest IC50 (1325.03 and 468.15 µg/mL, respectively). This study highlights the potential effects of chestnut shells on OM.

Natural Products for the Prevention and Treatment of Oral Mucositis-A Review.

Cancer, a major world public health problem, is associated with chemotherapy treatments whose administration leads to secondary concerns, such as oral mucositis (OM). The OM disorder is characterized by the presence of ulcers in the oral mucosa that cause pain, bleeding, and difficulty in ingesting fluids and solids, or speaking. Bioactive compounds from natural sources have arisen as an effective approach for OM. This review aims to summarize the new potential application of different natural products in the prevention and treatment of OM in comparison to conventional ones, also providing a deep insight into the most recent clinical studies. Natural products, such as Aloe vera, Glycyrrhiza glabra, Camellia sinensis, Calendula officinalis, or honeybee crops, constitute examples of sources of bioactive compounds with pharmacological interest due to their well-reported activities (e.g., antimicrobial, antiviral, anti-inflammatory, analgesic, or wound healing). These activities are associated with the bioactive compounds present in their matrix (such as flavonoids), which are associated with in vivo biological activities and minimal or absent toxicity. Finally, encapsulation has arisen as a future opportunity to preserve the chemical stability and the drug bioa vailability of bioactive compounds and, most importantly, to improve the buccal retention period and the therapeutic effects.

Draft Genome Sequence of a Leptospira kirschneri Serovar Mozdok Type 2 Strain Isolated from a Horse in Portugal.

Leptospira kirschneri is an agent causing leptospirosis in animals and humans. We report the draft genome sequence of Leptospira kirschneri serovar Mozdok type 2 strain Horse 112, comprising 485 contigs and having a genome size of 4,301,784 bp. This genome will facilitate studying important mechanisms for clinical outcomes.

Sex-specific changes in peripheral metabolism in a model of chronic anxiety induced by prenatal stress.

BACKGROUND: Prenatal stress is associated with increased susceptibility to psychiatric and metabolic disorders later in life. Prenatal exposure to stress mediators may have sex-dependent effects on offspring brain and metabolic function, promoting a sex-specific vulnerability to psychopathology and metabolic alterations at adulthood. In this work, the impact of prenatal stress on glucose homeostasis and peripheral metabolism of male and female offspring was investigated in a chronic anxiety animal model. METHODS: Pregnant Wistar rats were injected with saline or glucocorticoid (dexamethasone: 1 mg/kg, subcutaneous) at gestational days 18 and 19. Male and female offspring weight was monitored, and anxious-like behaviour and peripheral insulin-sensitive tissues were analysed at adulthood. RESULTS: At birth, females and males prenatally exposed to stress presented decreased body weight which remained low in females. At adulthood, a morphological disorganization of the Langerhans islets was observed in both sexes prenatally exposed to stress, yet not changes in insulin levels were detected. Also, prenatal stress increased glucose transporter 4 (GLUT-4) levels in female and male adipose tissues and decreased insulin receptor levels in the liver and skeleton muscle but only in females. CONCLUSIONS: Exposure to stress mediators in critical periods of development negatively affects behaviour and metabolism. Prenatal stress programmes offspring peripheral metabolism in a sex-specific manner, emphasizing that the response to stress in critical periods of development may be sex-specific having each sex different vulnerabilities to psychiatric and metabolic disorders. Considering sex-specificities may provide critical clues for the design of preventive strategies and for early therapeutic intervention.

Revisiting Ionic Liquid Structure-Property Relationship: A Critical Analysis.

In the last few years, ionic liquids (ILs) have been the focus of extensive studies concerning the relationship between structure and properties and how this impacts their application. Despite a large number of studies, several topics remain controversial or not fully answered, such as: the existence of ion pairs, the concept of free volume and the effect of water and its implications in the modulation of ILs physicochemical properties. In this paper, we present a critical review of state-of-the-art literature regarding structure-property relationship of ILs, we re-examine analytical theories on the structure-property correlations and present new perspectives based on the existing data. The interrelation between transport properties (viscosity, diffusion, conductivity) of IL structure and free volume are analysed and discussed at a molecular level. In addition, we demonstrate how the analysis of microscopic features (particularly using NMR-derived data) can be used to explain and predict macroscopic properties, reaching new perspectives on the properties and application of ILs.

Occupationally Acquired Q Fever in Shepherds and Sheep Milk Cheesemakers.

Q fever is a zoonosis caused by Coxiella burnetii, and transmission to humans is often associated with contact with ovine and caprine livestock. Those exposed to sheep are particularly at high risk of infection. Recent studies show that Q fever is increasing in sheep farms in Portugal raising alerts on spillover to humans. We detected anti-C. burnetii IgG in shepherds and sheep milk cheesemakers (27 [28.1%] in a total of 96; 95% confidence interval [CI] 19.4-38.2%) and in controls (21 [8.1%] in a total of 260; 95% CI 5.1-12.1%), pointing to an increased risk of C. burnetii infection (P = 0.0001), with an odds ratio for anti-C. burnetii of 4.45 (95% CI 2.4-8.4%; P = 0.0001), in individuals with occupational contact with sheep in Portugal.

Impact on mortality of direct admission versus interhospital transfer in patients with ST-segment elevation myocardial infarction undergoing primary percutaneous coronary intervention. / Impacto na mortalidade da admissão direta versus transferência inter­hospitalar nos doentes com enfarte agudo do miocárdio com elevação do segmento ST submetidos a intervenção coronária percutânea primária.

INTRODUCTION: In a primary percutaneous coronary intervention (PCI) program, interhospital transfer of patients with ST-elevation myocardial infarction (STEMI) can increase ischemic time, compared to patients who are admitted directly to a catheterization laboratory. OBJECTIVES: To assess the impact of interhospital transfer in patients with STEMI undergoing primary PCI, in terms of time to reperfusion and one-year mortality. METHODS: This was an observational, retrospective, longitudinal study of patients with STEMI admitted to Hospital de Braga between June 2011 and May 2016, who were treated successfully within 12 hours of symptom onset. A total of 1222 patients were included and divided into two groups according to admission to Hospital de Braga: direct or interhospital transfer. RESULTS: In this study, 37.0% (n=452) of the population were admitted directly to Hospital de Braga and 63.0% (n=770) were transferred from other hospitals. Although timings (in min) until reperfusion were longer in interhospital transfer patients (symptom onset-first medical contact (median 76.5, IQR 40.3-150 vs. 91.0, IQR 50-180, p=0.002), first medical contact-reperfusion (median 87.5, IQR 69.0-114 vs. 145, IQR 115-199, p<0.001) and symptom onset-reperfusion (median 177, IQR 125-265 vs. 265, IQR 188-400, p<0.001)), one-year mortality did not differ significantly between the groups (53 [11.7%] vs. 71 [9.2%], p=0.193). In multivariate analysis, age, symptom onset-reperfusion time and especially Killip class IV at admission (HR 11.2, 95% CI 6.35-19.8, p<0.001) were the main independent predictors of one-year mortality. CONCLUSION: Interhospital transfer of patients with STEMI increased the time before PCI. No differences were detected between groups in one-year mortality. This may be related to the fact that the direct admission group had twice as many patients in Killip class IV as the interhospital transfer group.

The Post-Dictatorship Memory Politics in Portugal Which Erased Political Violence from the Collective Memory.

Former clandestine militants' voices and stories have been recurrently silenced in the Portuguese "battle over memory", because their activities were linked to events, such as the Revolution of 25 April 1974, which have themselves been politically and socially depreciated in mainstream political narratives. Only recently did the traditional political narratives start to be questioned and debated by Portuguese scholars. Such political narratives took root in the country in the decades that followed the April Revolution, with various scholars and politicians denying the fascist categorisation of Estado Novo and adopting an authoritarian, non-totalitarian and non-fascist perspective, while recurrently depicting the Revolution as highly negative (namely as the source of the economic troubles of the country). Thus, for a long time, Portuguese conservatives opted to avoid debates on the 48 years of the Estado Novo's regime which, among other things, maintained a very repressive and violent political police force, a camp of forced labour in Cape Vert known as Tarrafal, and a Colonial War on three African fronts. This article examines the existent academic publications which counter such oblivion of memory regarding armed struggle in Portugal. It also explores the reasons behind, on the one hand, the whitewashing of Estado Novo and the historical revisionism typical of the 1970s and 1980s, and, on the other hand, the "rebellion of memory" which emerged in the 1990s.

Prospective Serosurvey of Coxiella burnetii Antibodies in Selected Sheep of Portugal.

Q fever is a zoonotic disease caused by Coxiella burnetii that is highly prevalent across the world. In this study, a prospective serosurvey was performed to study C. burnetii circulation in a population of sheep in the central region of Portugal. Blood from a representative sample of 168 animals was drawn in both 2015 and 2016, and sera were tested for IgG anti-C. burnetii by EIA. In 2015, 7.7% (13/168) animals tested positive for IgG anti-C. burnetii, while in 2016, 17.3% (29/168) tested positive, showing a statistically significant (P = 0.008) increase in anti-C. burnetii seroprevalence. Results support the notion that Q fever is emerging in central Portugal.

Natural variations in the biofilm-associated protein BslA from the genus Bacillus.

BslA is a protein secreted by Bacillus subtilis which forms a hydrophobic film that coats the biofilm surface and renders it water-repellent. We have characterised three orthologues of BslA from Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus pumilus as well as a paralogue from B. subtilis called YweA. We find that the three orthologous proteins can substitute for BslA in B. subtilis and confer a degree of protection, whereas YweA cannot. The degree to which the proteins functionally substitute for native BslA correlates with their in vitro biophysical properties. Our results demonstrate the use of naturally-evolved variants to provide a framework for teasing out the molecular basis of interfacial self-assembly.

Bifunctionality of a biofilm matrix protein controlled by redox state.

Biofilms are communities of microbial cells that are encapsulated within a self-produced polymeric matrix. The matrix is critical to the success of biofilms in diverse habitats; however, many details of the composition, structure, and function remain enigmatic. Biofilms formed by the Gram-positive bacterium Bacillus subtilis depend on the production of the secreted film-forming protein BslA. Here, we show that a gradient of electron acceptor availability through the depth of the biofilm gives rise to two distinct functional roles for BslA and that these roles can be genetically separated through targeted amino acid substitutions. We establish that monomeric BslA is necessary and sufficient to give rise to complex biofilm architecture, whereas dimerization of BslA is required to render the community hydrophobic. Dimerization of BslA, mediated by disulfide bond formation, depends on two conserved cysteine residues located in the C-terminal region. Our findings demonstrate that bacteria have evolved multiple uses for limited elements in the matrix, allowing for alternative responses in a complex, changing environment.

Spermidine promotes Bacillus subtilis biofilm formation by activating expression of the matrix regulator slrR.

Ubiquitous polyamine spermidine is not required for normal planktonic growth of Bacillus subtilis but is essential for robust biofilm formation. However, the structural features of spermidine required for B. subtilis biofilm formation are unknown and so are the molecular mechanisms of spermidine-stimulated biofilm development. We report here that in a spermidine-deficient B. subtilis mutant, the structural analogue norspermidine, but not homospermidine, restored biofilm formation. Intracellular biosynthesis of another spermidine analogue, aminopropylcadaverine, from exogenously supplied homoagmatine also restored biofilm formation. The differential ability of C-methylated spermidine analogues to functionally replace spermidine in biofilm formation indicated that the aminopropyl moiety of spermidine is more sensitive to C-methylation, which it is essential for biofilm formation, but that the length and symmetry of the molecule is not critical. Transcriptomic analysis of a spermidine-depleted B. subtilis speD mutant uncovered a nitrogen-, methionine-, and S-adenosylmethionine-sufficiency response, resulting in repression of gene expression related to purine catabolism, methionine and S-adenosylmethionine biosynthesis and methionine salvage, and signs of altered membrane status. Consistent with the spermidine requirement in biofilm formation, single-cell analysis of this mutant indicated reduced expression of the operons for production of the exopolysaccharide and TasA protein biofilm matrix components and SinR antagonist slrR Deletion of sinR or ectopic expression of slrR in the spermidine-deficient ΔspeD background restored biofilm formation, indicating that spermidine is required for expression of the biofilm regulator slrR Our results indicate that spermidine functions in biofilm development by activating transcription of the biofilm matrix exopolysaccharide and TasA operons through the regulator slrR.

A comparison between pure active pharmaceutical ingredients and therapeutic deep eutectic solvents: Solubility and permeability studies.

THEDES, so called therapeutic deep eutectic solvents are here defined as a mixture of two components, which at a particular molar composition become liquid at room temperature and in which one of them is an active pharmaceutical ingredient (API). In this work, THEDES based on menthol complexed with three different APIs, ibuprofen (ibu), BA (BA) and phenylacetic acid (PA), were prepared. The interactions between the components that constitute the THEDES were studied by NMR, confirming that the eutectic system is formed by H-bonds between menthol and the API. The mobility of the THEDES components was studied by PFGSE NMR spectroscopy. It was determined that the self-diffusion of the species followed the same behavior as observed previously for ionic liquids, in which the components migrate via jumping between voids in the suprastructure created by punctual thermal fluctuations. The solubility and permeability of the systems in an isotonic solution was evaluated and a comparison with the pure APIs was established through diffusion and permeability studies carried out in a Franz cell. The solubility of the APIs when in the THEDES system can be improved up to 12 fold, namely for the system containing ibu. Furthermore, for this system the permeability was calculated to be 14×10-5cm/s representing a 3 fold increase in comparison with the pure API. With the exception of the systems containing PA an increase in the solubility, coupled with an increase in permeability was observed. In this work, we hence demonstrate the efficiency of THEDES as a new formulation for the enhancement of the bioavailability of APIs by changing the physical state of the molecules from a solid dosage to a liquid system.

Direct detection and differentiation of pathogenic Leptospira species using a multi-gene targeted real time PCR approach.

Leptospirosis is a growing public and veterinary health concern caused by pathogenic species of Leptospira. Rapid and reliable laboratory tests for the direct detection of leptospiral infections in animals are in high demand not only to improve diagnosis but also for understanding the epidemiology of the disease. In this work we describe a novel and simple TaqMan-based multi-gene targeted real-time PCR approach able to detect and differentiate Leptospira interrogans, L. kirschneri, L. borgpeteresenii and L. noguchii, which constitute the veterinary most relevant pathogenic species of Leptospira. The method uses sets of species-specific probes, and respective flanking primers, designed from ompL1 and secY gene sequences. To monitor the presence of inhibitors, a duplex amplification assay targeting both the mammal ß-actin and the leptospiral lipL32 genes was implemented. The analytical sensitivity of all primer and probe sets was estimated to be <10 genome equivalents (GE) in the reaction mixture. Application of the amplification reactions on genomic DNA from a variety of pathogenic and non-pathogenic Leptospira strains and other non-related bacteria revealed a 100% analytical specificity. Additionally, pathogenic leptospires were successfully detected in five out of 29 tissue samples from animals (Mus spp., Rattus spp., Dolichotis patagonum and Sus domesticus). Two samples were infected with L. borgpetersenii, two with L. interrogans and one with L. kirschneri. The possibility to detect and identify these pathogenic agents to the species level in domestic and wildlife animals reinforces the diagnostic information and will enhance our understanding of the epidemiology of leptopirosis.

Genotypic diversity of environmental Cryptococcus neoformans isolates from Northern Portugal.

The Cryptococcus neoformans/C. gattii species complex members are the main agents of systemic cryptococcosis. This disease is believed to be acquired from the environment via fungal cell inhalation. Often, isolates recovered from environmental and clinical sources have proven to be genotypically similar. We assessed the occurrence of C. neoformans and C. gattii in environmental substrates collected in a Portuguese region. Twenty-eight isolates were identified as C. neoformans - five from decaying Eucalyptus leaves and 23 from domestic pigeon droppings. The isolates were genotyped using a URA5-RFLP approach. The C. neoformans VNIV (53.6%, n = 15) and VNI (32.1%, n = 9) genotypes were abundantly present among environmental isolates. The hybrid VNIII (14.3%, n = 4) genotype was underrepresented and the VNII was not found. Cryptococcus gattii was also not found although some isolates yielded a positive canavanine-glycine-bromothymol blue test.

Alkyl-glycerol rescues plasmalogen levels and pathology of ether-phospholipid deficient mice.

A deficiency of plasmalogens, caused by impaired peroxisomal metabolism affects normal development and multiple organs in adulthood. Treatment options aimed at restoring plasmalogen levels may be relevant for the therapy of peroxisomal and non-peroxisomal disorders. In this study we determined the in vivo efficacy of an alkyl glycerol (AG), namely, 1-O-octadecyl-rac-glycerol, as a therapeutic agent for defects in plasmalogen synthesis. To achieve this, Pex7 knockout mice, a mouse model for Rhizomelic Chondrodysplasia Punctata type 1 characterized by the absence of plasmalogens, and WT mice were fed a control diet or a diet containing 2% alkyl-glycerol. Plasmalogen levels were measured in target organs and the biochemical data were correlated with the histological analysis of affected organs. Plasmalogen levels in all peripheral tissues of Pex7 KO mice fed the AG diet for 2 months normalized to the levels of AG fed WT mice. In nervous tissues of Pex7 KO mice fed the AG-diet, plasmalogen levels were significantly increased compared to control fed KO mice. Histological analysis of target organs revealed that the AG-diet was able to stop the progression of the pathology in testis, adipose tissue and the Harderian gland. Interestingly, the latter tissues are characterized by the presence of lipid droplets which were absent or reduced in size and number when ether-phospholipids are lacking, but which can be restored with the AAG treatment. Furthermore, nerve conduction in peripheral nerves was improved. When given prior to the occurrence of major pathological changes, the AG-diet prevented or ameliorated the pathology observed in Pex7 KO mice depending on the degree of plasmalogen restoration. This study provides evidence of the beneficial effects of treating a plasmalogen deficiency with alkyl-glycerol.