The Relationship between Photoluminescence Emissions and Photocatalytic Activity of CeO2 Nanocrystals.

In this work, we focus on understanding the morphology and photocatalytic properties of CeO2 nanocrystals (NCs) synthesized via a microwave-assisted solvothermal method using acetone and ethanol as solvents. Wulff constructions reveal a complete map of available morphologies and a theoretical-experimental match with octahedral nanoparticles obtained through synthesis using ethanol as solvent. NCs synthesized in acetone show a greater contribution of emission peaks in the blue region (∼450 nm), which may be associated with higher Ce3+ concentration, originating shallow-level defects within the CeO2 lattice while for the samples synthesized in ethanol a strong orange-red emission (∼595 nm) suggests that oxygen vacancies may originate from deep-level defects within the optical bandgap region. The superior photocatalytic response of CeO2 synthesized in acetone compared to that of CeO2 synthesized in ethanol may be associated with an increase in long-/short-range disorder within the CeO2 structure, causing the Egap value to decrease, facilitating light absorption. Furthermore, surface (100) stabilization in samples synthesized in ethanol may be related to low photocatalytic activity. Photocatalytic degradation was facilitated by the generation of ·OH and ·O2- radicals as corroborated by the trapping experiment. The mechanism of enhanced photocatalytic activity has been proposed suggesting that samples synthesized in acetone tend to have lower e'─h· pair recombination, which is reflected in their higher photocatalytic response.

New objective measurements of semen wave motion are associated with fertility in sheep.

In sheep, wave motion in semen is currently used by AI centres to select ejaculates for insemination. Despite its low cost, convenience and established ability to predict fertility, the subjectivity of this assessment is a limiting factor for its applicability. The aims of the present study were to establish an objective method for the analysis of wave motion and to assess the associations of objective parameters with fertility after cervical insemination. Collective sperm motion in undiluted semen was observed by phase contrast microscopy at low magnification in a 100-µm deep glass chamber. Images of moving dark waves over a grey background were recorded and analysed by the optic flow method, producing several velocity-related parameters. Turbulence was assessed from the motion of fluorescent polystyrene beads. Among objective parameters, optical flow entropy and the average speed of beads were both able to discriminate ejaculates suitable for insemination. Two synthetic variables of optic flow and bead motion and a global objective variable were computed from linear combinations of individual parameters and compared with the subjective motion score for their predictive value. These were as efficient as the wave motion score for assessing fertility and can be proposed for the assessment of ram semen in routine AI procedures.

Geriatric nutritional risk index as a nutritional and survival risk assessment tool in stable outpatients with systolic heart failure.

BACKGROUND AND AIMS: Malnutrition is frequent in heart failure (HF). However, the best tool for evaluating malnutrition in geriatric patients with HF with reduced ejection fraction (HFrEF) is unknown. This study aimed to evaluate the incremental prognostic value of the geriatric nutritional risk index (GNRI) in stable geriatric outpatients with HFrEF compared with a clinical/laboratory prognostic model. METHODS AND RESULTS: A total of 143 outpatients with HFrEF, aged >65 years, a LVEF <40%, and who were stable and on optimal therapy were studied. Follow-up lasted 3 years. The outcome was all-cause death. The GNRI was calculated as follows: [(1.489 × serum albumin (g/L)) + (41.7 × (current body weight/ideal weight)]. The 3-year death rate was 36.4% and 16 (11.2%) patients were at risk of malnutrition (GNRI ≤98). Deceased patients had a lower GNRI (113.6 ± 9.1 vs. 105.6 ± 9.2; p < 0.001) than did survivors. Greater values of the GNRI (hazard ratio = 0.93, 95% confidence interval [CI] = 0.90-0.95; p < 0.001) and GNRI >98 (hazard ratio = 0.29, 95% CI 0.15-0.57; p < 0.001) were associated with better survival. These factors remained significant after adjustment of significant confounders. The GNRI was a better discriminator of death than weight and albumin. Adding the GNRI to the clinical/laboratory predictor survival model significantly increased the c-statistics from 0.93 to 0.95 (p < 0.001) and the chi-square likelihood ratio test from 106.15 to 119.9. CONCLUSION: The risk of malnutrition, as assessed by the GNRI, in stable geriatric outpatients with HFrEF is a strong independent predictor of survival. The GNRI adds significant prognostic information to the clinical/laboratory model.

Influence of bethanechol on salivary parameters in irradiated patients.

BACKGROUND: Some studies have shown evidence that the prophylactic use of bethanechol chloride (BC) may be useful in preventing the incidence and/or severity of xerostomia (XT). However, the indication of BC in irradiated patients with XT needs to be better characterized. The study aimed to evaluate the influence of BC on XT, salivary flow rate, and salivary composition in patients previously submitted to head and neck radiotherapy. MATERIAL AND METHODS: Forty five irradiated patients complaining of XT used 50 mg/day of BC for 3 months, and the salivary parameters were evaluated in 4 Phases (Before BC therapy, after one month of BC, 2 months of BC, and 3 months of BC). Biochemical analysis included buffering capacity; pH; total protein concentration (TP); amylase concentration (AM); catalase (CAT) and peroxidase (PX) activities. In addition, unstimulated and stimulated salivary flow rates were determined and XT was classified. RESULTS: According to the XT grading system used, patients showed improvement in XT between Phase 1, and Phases 2, 3 and 4. In addition, some changes were observed in TP concentration (decreased); AM concentration (increased); and PX and CAT activities (decreased and increased, respectively) after Phase 2, for stimulated saliva collection (p<0.05). CONCLUSIONS: Our results suggested that when BC was used to treat salivary gland dysfunction induced by head and neck radiotherapy, improvement in XT symptoms, and some changes in saliva composition were shown.

Glu311 and Arg337 Stabilize a Closed Active-site Conformation and Provide a Critical Catalytic Base and Countercation for Green Bioluminescence in Beetle Luciferases.

Beetle luciferases elicit the emission of different bioluminescence colors from green to red. Whereas firefly luciferases emit yellow-green light and are pH-sensitive, undergoing a typical red-shift at acidic pH and higher temperatures and in the presence of divalent heavy metals, click beetle and railroadworm luciferases emit a wider range of colors from green to red but are pH-independent. Despite many decades of study, the structural determinants and mechanisms of bioluminescence colors and pH sensitivity remain enigmatic. Here, through modeling studies, site-directed mutagenesis, and spectral and kinetic studies using recombinant luciferases from the three main families of bioluminescent beetles that emit different colors of light (Macrolampis sp2 firefly, Phrixotrix hirtus railroadworm, and Pyrearinus termitilluminans click beetle), we investigated the role of E311 and R337 in bioluminescence color determination. All mutations of these residues in firefly luciferase produced red mutants, indicating that the preservation of opposite charges and the lengths of the side chains of E311 and R337 are essential for keeping a salt bridge that stabilizes a closed hydrophobic conformation favorable for green light emission. Kinetic studies indicate that residue R337 is important for binding luciferin and creating a positively charged environment around excited oxyluciferin phenolate. In Pyrearinus green-emitting luciferase, the R334A mutation causes a 27 nm red-shift, whereas in Phrixotrix red-emitting luciferase, the L334R mutation causes a blue-shift that is no longer affected by guanidine. These results provide compelling evidence that the presence of arginine at position 334 is essential for blue-shifting the emission spectra of most beetle luciferases. Therefore, residues E311 and R337 play both structural and catalytic roles in bioluminescence color determination, by stabilizing a closed hydrophobic conformation favorable for green light emission, and also providing a base to accept excited oxyluciferin phenol proton, and a countercation to shield the negative charge of E311 and to stabilize excited oxyluciferin phenolate, blue-shifting emission spectra in most beetle luciferases.

Self-declared ethnicity and genomic ancestry in prostate cancer patients from Brazil.

Some studies of polymorphisms in prostate cancer (PCa) analyze individuals in a uniform manner, regardless of genetic ancestry. However, PCa aggressiveness differs between subjects of African descent and those of European extraction. Thus, genetic ancestry analysis may be used to detect population stratification in case-control association studies. We genotyped 11 ancestry informative markers to estimate the contributions of African, European, and Amerindian ancestries in a case-control sample of 213 individuals from Bahia State, Northeast Brazil, including 104 PCa patients. We compared this data with self-reported ancestry and the stratification of cases by PCa aggressiveness according to Gleason score. A larger African genetic contribution (44%) was detected among cases, and a greater European contribution (61%) among controls. Self-declaration data revealed that 74% of PCa patients considered themselves non-white (black and brown), and 41.3% of controls viewed themselves as white. Our data showed a higher degree of European ancestry among fast-growing cancer cases than those of intermediate and slow development. This differs from many previous studies, in which the prevalence of African ancestry has been reported for all grades. Differences were observed between degrees of PCa aggressiveness in terms of genetic ancestry. In particular, the greater European contribution among patients with high-grade PCa indicates that a population's genetic structure can influence case-control studies. This investigation contributes to our understanding of the genetic basis of tumor aggressiveness among groups of different genetic ancestries, especially admixed populations, and has significant implications for the assessment of inter-population heterogeneity in drug treatment effects.

Ion release and mechanical properties of calcium silicate and calcium hydroxide materials used for pulp capping.

AIM: To compare the ion release and mechanical properties of a calcium hydroxide (Dycal) and two calcium silicate (MTA Angelus and Biodentine) cements. METHODOLOGY: Calcium and hydroxyl ion release in water from 24-h set cements were calculated from titration with HCl (n = 3). Calcium release after 7, 14, 21 and 28 days at pH 5.5 and 7.0 was measured using ICP-OES (n = 6). Flexural strength (FS) and modulus (E) were tested after 48-h storage, and compressive strength (CS) was tested after 48 h and 7 days (n = 10). Ion release and mechanical data were subjected to anova/Tukey and Kruskal-Wallis/Mann-Whitney tests, respectively (α = 0.05). RESULTS: Titration curves revealed that Dycal released significantly fewer ions in solution than calcium silicates (P < 0.001). Calcium release remained constant at pH 7.0, whilst at pH 5.5, it dropped significantly by 24% after 21 days (P < 0.05). At pH 5.5, MTA Angelus released significantly more calcium than Dycal (P < 0.01), whilst Biodentine had superior ion release than Dycal at pH 7.0 (P < 0.01). Biodentine had superior flexural strength, flexural modulus and compressive strength than the other cements, whilst MTA Angelus had higher modulus than Dycal (P < 0.001). CONCLUSIONS: Immediate calcium and hydroxyl ion release in solution was significantly lower for Dycal. In general, all materials released constant calcium levels over 28 days, but release from Dycal was significantly lower than Biodentine and MTA Angelus depending on pH conditions. Biodentine had substantially higher strength and modulus than MTA Angelus and Dycal, both of which demonstrated low stress-bearing capabilities.

Characteristics of Haemophilus influenzae invasive isolates from Portugal following routine childhood vaccination against H. influenzae serotype b (2002-2010).

We aimed to characterize Haemophilus influenzae invasive isolates recovered in Portugal over a 9-year period (2002-2010) following the inclusion of H. influenzae serotype b (Hib) conjugate vaccination in the National Immunization Program (NIP) in the year 2000 and compare the results with those obtained in a similar study from the pre-vaccination era (1989-2001) previously described by us. As part of a laboratory-based passive surveillance system, 144 invasive isolates obtained in 28 Portuguese hospitals were received at the National Reference Laboratory for Bacterial Respiratory Infections and were characterized. Capsular types and antibiotic susceptibility patterns were determined. The ftsI gene encoding PBP3 was sequenced for ß-lactamase-negative ampicillin-resistant (BLNAR) isolates. Genetic relatedness among isolates was examined by multilocus sequencing typing (MLST). Most isolates (77.1%) were non-capsulated, a significant increase compared to the pre-vaccination era (19.0%, p < 0.001). Serotype b strains decreased significantly (from 81.0 to 13.2%, p < 0.001) and serotype f increased significantly (from 0.8 to 6.9%, p = 0.03). Ten percent of the isolates were ß-lactamase producers, a value lower than that previously observed (26.9%, p = 0.005). Eight percent of all isolates were BLNAR. A high genetic diversity among non-capsulated isolates was found. By contrast, capsulated isolates were clonal. The implementation of Hib vaccination has resulted in a significant decline in the proportion of serotype b H. influenzae invasive disease isolates. Most episodes of invasive disease occurring in Portugal are now due to fully susceptible, highly diverse, non-capsulated strains. Given the evolving dynamics of this pathogen and the increase in non-type b capsulated isolates, continuous surveillance is needed.

Genomic insights into the expansion of carbapenem-resistant Klebsiella pneumoniae within Portuguese hospitals.

Carbapenem-resistant Klebsiella pneumoniae (CR-KP) are a public health concern, causing infections with a high mortality rate, limited therapeutic options and challenging infection control strategies. In Portugal, the CR-KP rate has increased sharply, but the factors associated with this increase are poorly explored. In order to address this question, phylogenetic and resistome analysis were used to compare the draft genomes of 200 CR-KP isolates collected in 2017-2019 from five hospitals in the Lisbon region, Portugal. Most CR-KP belonged to sequence type (ST) 13 (29%), ST17 (15%), ST348 (13%), ST231 (12%) and ST147 (7%). Carbapenem resistance was conferred mostly by the presence of KPC-3 (74%) or OXA-181 (18%), which were associated with IncF/IncN and IncX plasmids, respectively. Almost all isolates were multi-drug resistant, harbouring resistance determinants to aminoglycosides, beta-lactams, trimethoprim, fosfomycin, quinolones and sulphonamides. In addition, 11% of isolates were resistant to colistin. Colonizing and infecting isolates were highly related, and most colonized patients (89%) reported a previous hospitalization. Moreover, among the 171 events of cross-dissemination identified by core genome multi-locus sequence typing data analysis (fewer than five allelic differences), 41 occurred between different hospitals and 130 occurred within the same hospital. The results suggest that CR-KP dissemination in the Lisbon region results from acquisition of carbapenemases in mobile genetic elements, influx of CR-KP into the hospitals by colonized ambulatory patients, and transmission of CR-KP within and between hospitals. Prudent use of carbapenems, patient screening at hospital entry, and improvement of infection control are needed to decrease the burden of CR-KP infection in Portugal.

Genetic diversity of the HLA-G coding region in Amerindian populations from the Brazilian Amazon: a possible role of natural selection.

HLA-G has an important role in the modulation of the maternal immune system during pregnancy, and evidence that balancing selection acts in the promoter and 3'UTR regions has been previously reported. To determine whether selection acts on the HLA-G coding region in the Amazon Rainforest, exons 2, 3 and 4 were analyzed in a sample of 142 Amerindians from nine villages of five isolated tribes that inhabit the Central Amazon. Six previously described single-nucleotide polymorphisms (SNPs) were identified and the Expectation-Maximization (EM) and PHASE algorithms were used to computationally reconstruct SNP haplotypes (HLA-G alleles). A new HLA-G allele, which originated in Amerindian populations by a crossing-over event between two widespread HLA-G alleles, was identified in 18 individuals. Neutrality tests evidenced that natural selection has a complex part in the HLA-G coding region. Although balancing selection is the type of selection that shapes variability at a local level (Native American populations), we have also shown that purifying selection may occur on a worldwide scale. Moreover, the balancing selection does not seem to act on the coding region as strongly as it acts on the flanking regulatory regions, and such coding signature may actually reflect a hitchhiking effect.

The luciferin binding site residues C/T311 (S314) influence the bioluminescence color of beetle luciferases through main-chain interaction with oxyluciferin phenolate.

Beetle luciferases emit different bioluminescence colors from green to red; however, no clear relationship between the identity of the luciferin binding site residues and bioluminescence colors was found in different luciferases, and it is unclear whether critical interactions affecting emission spectra occur on the thiazolyl or on the benzothiazolyl sides of the luciferin binding site. Through homology modeling and site-directed mutagenesis using our multicolor set of beetle luciferases (Pyrearinus termitilluminans larval click beetle, Pte, λ(max) = 534 nm; Phrixothrix hirtus railroad worm red emitting, PxRE, λ(max) = 623 nm; and Macrolampis sp2 firefly, Mac, λ(max) = 564 nm), we show that the residues C/T311 (S314) play an important role in bioluminescence color determination. Modeling studies indicate that the main-chain carbonyls of these residues are close to both oxyluciferin phenolate and AMP, whereas the side chains pack against second-shell residues. The C311(S314)A mutation considerably red shifts the spectra of the green-yellow-emitting luciferases (Pte λ(max) = 534 to 590 nm; Mac λ(max) = 564 to 583/613 nm) and affects the K(M) values for luciferin and ATP, but not the spectrum of the red-emitting luciferase. On the other hand, whereas the exchange between C/T311 (S314) caused smaller effects on the emission spectra of green-yellow-emitting luciferases, the C311T substitution (naturally found in green-emitting railroad worm luciferases) resulted in the largest reported blue shift in P. hirtus red-emitting luciferase (λ(max) = 623 to 606 nm). Altogether, these results indicate that the stability of residues C/T311 (S314) and the size of the cavity around oxyluciferin phenolate affect bioluminescence colors and suggest, for the first time, the occurrence of a critical interaction between main-chain carbonyls of position 311 (314) residues and oxyluciferin phenolate.

Functional morphology and wasp pollination of two South American asclepiads (Asclepiadoideae-Apocynaceae).

UNLABELLED: BACKGROUND AND AIMS The extreme complexity of asclepiad flowers (Asclepiadoideae-Apocynaceae) has generated particular interest in the pollination biology of this group of plants especially in the mechanisms involved in the pollination processes. This study compares two South American species, Morrenia odorata and Morrenia brachystephana, with respect to morphology and anatomy of flower structures, dynamic aspects of the pollination mechanism, diversity of visitors and effectiveness of pollinators. METHODS: Floral structure was studied with fresh and fixed flowers following classical techniques. The pollination mechanism was studied by visiting fresh flowers in the laboratory with artificial pollinator body parts created with an eyelash. Morphometric and nectar measurements were also taken. Pollen transfer efficiency in the flowers was calculated by recording the frequency of removed and inserted pollinia. Visitor activity was recorded in the field, and floral visitors were captured for subsequent analysis of pollen loads. Finally, pollinator effectiveness was calculated with an index. KEY RESULTS: The detailed structure of the flowers revealed a complex system of guide rails and chambers precisely arranged in order to achieve effective pollinaria transport. Morrenia odorata is functionally specialized for wasp pollination, and M. brachystephana for wasp and bee pollination. Pollinators transport chains of pollinaria adhered to their mouthparts. CONCLUSIONS: Morrenia odorata and M. brachystephana present differences in the morphology and size of their corona, gynostegium and pollinaria, which explain the differences in details of the functioning of the general pollination mechanism. Pollination is performed by different groups of highly effective pollinators. Morrenia species are specialized for pollination mainly by several species of wasps, a specialized pollination which has been poorly studied. In particular, pompilid wasps are reported as important pollinators in other regions outside South Africa. A putative new function of nectar in asclepiads is presented, as it would be contributing to the pollination mechanism.

Synthesis and defect characterization of hybrid ceria nanostructures as a possible novel therapeutic material towards COVID-19 mitigation.

This study reports the synthesis of hybrid nanostructures composed of cerium dioxide and microcrystalline cellulose prepared by the microwave-assisted hydrothermal route under distinct temperature and pH values. Their structural, morphological and spectroscopic behaviors were investigated by X-Rays Diffraction, Field Emission Gun Scanning Electron Microscopy, High-Resolution Transmission Electron Microscopy, and Fourier-Transform Infrared, Ultraviolet-Visible, Raman and Positron Annihilation Lifetime spectroscopies to evaluate the presence of structural defects and their correlation with the underlying mechanism regarding the biocide activity of the studied material. The samples showed mean crystallite sizes around 10 nm, characterizing the formation of quantum dots unevenly distributed along the cellulose surface with a certain agglomeration degree. The samples presented the characteristic Ce-O vibration close to 450 cm-1 and a second-order mode around 1050 cm-1, which is indicative of distribution of localized energetic levels originated from defective species, essential in the scavenging of reactive oxygen species. Positron spectroscopic studies showed first and second lifetime components ranging between 202-223 ps and 360-373 ps, respectively, revealing the presence of two distinct defective oxygen species, in addition to an increment in the concentration of Ce3+-oxygen vacancy associates as a function of temperature. Therefore, we have successfully synthesized hybrid nanoceria structures with potential multifunctional therapeutic properties to be further evaluated against the COVID-19.

Analysis of five polymorphic DNA markers for indirect genetic diagnosis of haemophilia A in the Brazilian population.

Hemophilia A is an X-linked, inherited, bleeding disorder caused by the partial or total inactivity of the coagulation factor VIII (FVIII). Due to difficulties in the direct recognition of the disease-associated mutation in the F8 gene, indirect diagnosis using polymorphic markers located inside or close to the gene is used as an alternative for determining the segregation of the mutant gene within families and thus for detecting carrier individuals and/or assisting in prenatal diagnosis. This study characterizes the allelic and haplotype frequencies, genetic diversity, population differentiation and linkage disequilibrium of five microsatellites (F8Int1, F8Int13, F8Int22, F8Int25.3 and IKBKG) in samples of healthy individuals from São Paulo, Rio Grande do Sul and Pernambuco and of patients from São Paulo with haemophilia A to determine the degree of informativeness of these microsatellites for diagnostic purposes. The interpopulational diversity parameters highlight the differences among the analyzed population samples. Regional differences in allelic frequencies must be taken into account when conducting indirect diagnosis of haemophilia A. With the exception of IKBKG, all of the microsatellites presented high heterozygosity levels. Using the markers described, diagnosis was possible in 10 of 11 families. The F8Int22, F8Int1, F8Int13, F8Int25.3 and IKBKG microsatellites were informative in seven, six, five and two of the cases, respectively, demonstrating the effectiveness of using these microsatellites in prenatal diagnosis and in carrier identification in the Brazilian population.

Signs for Early Detection of Dysphagia in Older Adults with Severe Alzheimer's Disease.

OBJECTIVES: The aim of this study was to detect signs of dysphagia in older adults with and without Alzheimer's disease (AD). STUDY DESIGN: A cross-sectional study. SETTING AND PARTICIPANTS: A total of 280 institutionalized older adults (with AD = 145; without AD = 135), aged > 70 years, with oral feeding, both sexes were examined. MEASUREMENTS: Modified water-swallowing test determined the dysphagia. Independent variables were dependency level (modified Barthel Index), oral health and function status (residual teeth, occlusal contacts, passive lip sealing, tongue function, rinsing and gargling ability), nutritional status (Body Mass Index, Mini Nutritional Assessment), and diet-related assessments (appetite, storing or stuffing food in the mouth). Statistical analysis was carried out by Fisher´s Exact and chi-square tests. The analyzes of associations with the dysphagia outcome were performed by simple and multiple logistic regression models. RESULTS: Severity of AD was significantly associated with dysphagia. The signs associated with dysphagia were decreased nutritional status, absence of appetite, and storing food in the mouth. CONCLUSION: These signs can be useful tools for early diagnosis of dysphagia in AD older adults.

Muon implantation experiments in films: Obtaining depth-resolved information.

Implanted positive muons with low energies (in the range 1-30 keV) are extremely useful local probes in the study of thin films and multi-layer structures. The average muon stopping depth, typically in the order of tens of nanometers, is a function of the muon implantation energy and of the density of the material, but the stopping range extends over a broad region, which is also in the order of tens of nanometers. Therefore, an adequate simulation procedure is required in order to extract the depth dependence of the experimental parameters. Here, we present a method to extract depth-resolved information from the implantation energy dependence of the experimental parameters in a low-energy muon spin spectroscopy experiment. The method and corresponding results are exemplified for a semiconductor film, Cu(In,Ga)Se2, covered with a thin layer of Al2O3, but can be applied to any heterostructure studied with low-energy muons. It is shown that if an effect is present in the experimental data, this method is an important tool to identify its location and depth extent.

Sequence analysis of 37 candidate genes for male infertility: challenges in variant assessment and validating genes.

BACKGROUND: The routine genetic analysis for diagnosing male infertility has not changed over the last twenty years, and currently available tests can only determine the etiology of 4% of unselected infertile patients. Thus, to create new diagnostic assays, we must better understand the molecular and genetic mechanisms of male infertility. Although next-generation sequencing allows for simultaneous analysis of hundreds of genes and the discovery of novel candidates related to male infertility, so far only a few gene candidates have enough sound evidence to support the gene-disease relationship. OBJECTIVE: Since complementary studies are required to validate genes, we aimed to analyze the presence of potentially pathogenic rare variants in a set of candidate genes related to azoospermia in a hitherto understudied South American population. SUBJECTS AND METHODS: We performed whole exome sequencing in a group of 16 patients with non-obstructive azoospermia from Ribeirão Preto, Brazil. Based on a recent systematic review of monogenic causes of male infertility, we selected a set of 37 genes related to azoospermia, Sertoli-Cell-Only histology, and spermatogenic arrest to analyze. The identified variants were confirmed by Sanger sequencing, and their functional consequence was predicted by in silico programs. RESULTS: We identified potential pathogenic variants in seven genes in six patients. Two variants, c.671A>G (p.(Asn224Ser)) in DMRT1 and c.91C>T (p.(Arg31Cys)) in REC8, have already been described in association with azoospermia. We also found new variants in genes that already have moderate evidence of being linked to spermatogenic failure (TEX15, KLHL10), in genes with limited evidence (DNMT3B, TEX14) and in one novel promising candidate gene that has no evidence so far (SYCE1L). DISCUSSION: Although this study included a small number of patients, the process of rationally selecting genes allowed us to detect rare potentially pathogenic variants, providing supporting evidence for validating candidate genes associated with azoospermia.

The proton and metal binding sites responsible for the pH-dependent green-red bioluminescence color tuning in firefly luciferases.

Firefly luciferases produce yellow-green light under physiological and alkaline conditions, however at acidic pH, higher temperatures or in the presence of heavy metals the color changes to red, a property called pH-sensitivity. Despite many decades of studies, the proton and metal binding sites responsible for pH-sensitivity remain enigmatic. Previously we suggested that the salt bridge E311/R337 keeps a closed conformation of the luciferin phenolate binding site. Here we further investigated the effect of this salt bridge and mutations of the neighbor residues H310 and E/N354, on metal and pH-sensitivity of firefly luciferases emitting distinct bioluminescence colors (Cratomorphus distinctus: 548 nm; Macrolampis sp2: 569 nm). The substitutions of H310 and E/N354 modulate metal sensitivity, whereas the carboxylate of E311 may work as the catalytic base essential for green bioluminescence and pH-sensitivity. Modeling studies showed that H310, E311 and E354 side-chains coordinate Zinc, constituting the metal binding site and the pH-sensor. Electrostatic potential and pKa calculations suggest that the external couple H310/E354 is affected by pH, whereas E311/R337 make a stabilized internal pair which retains excited oxyluciferin ejected proton near its phenolate group into a high energy state, promoting yellow-green bioluminescence. Protonation or metal binding weaken these electrostatic gates and their ability to retain the excited oxyluciferin released proton near its phenolate, promoting red light emission.

Participatory implementation of an antibiotic stewardship programme supported by an innovative surveillance and clinical decision-support system.

BACKGROUND: Antibiotic resistance will cause about 10 million deaths per year by 2050. Fighting antimicrobial resistance is a health priority. Interventions aimed to reduce antimicrobial resistance, such as antibiotic stewardship programmes (ASPs), must be implemented. To be effective, those interventions, and the implementation process, should be matched with social-cultural context. The complexity of ASPs can no longer be developed without considering both organizational and information systems. AIM: To support ASPs through the co-design and implementation, in collaboration with healthcare workers, of a surveillance and clinical decision-support system to monitor antibiotic resistance and improve antibiotic prescription. METHODS: The surveillance and clinical decision-support system was designed and implemented in three Portuguese hospitals, using a participatory approach between researchers and healthcare workers following the Design Science Research Methodology. FINDINGS: Based on healthcare workers' requirements, we developed HAITooL, a real-time surveillance and clinical decision-support system that integrates visualizations of patient, microbiology, and pharmacy data, facilitating clinical decision. HAITooL monitors antibiotic usage and rates of antibiotic-resistant bacteria, allowing early identification of outbreaks. It is a clinical decision-support tool that integrates evidence-based algorithms to support proper antibiotic prescription. HAITooL was considered valuable to support monitoring of antibiotic resistant infections and an important tool for ASP sustainability. CONCLUSION: ASP implementation can be leveraged through a surveillance and clinical decision-support system such as HAITooL that allows antibiotic resistance monitoring and supports antibiotic prescription, once it has been adapted to the context and specific needs of healthcare workers and hospitals.