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A simple and sensitive method based on a liquid droplet is described for the measurement of atmospheric ozone. A 30 µL drop of indigo blue solution is suspended in a flowing-air sampling stream. The ozone collected reacts with the indigo solution resulting in its decolorization. The colorimetric sensor is composed of two optical fibers and the source of monochromatic light was a red LED (625 nm). The calibration curve was constructed with ozone standard concentrations ranging from 37 - 123 ppbv. The detection limit achieved was 7.3 ppbv. The method considered here showed itself to be easy to apply with a fast response and a total analysis time of only 5 minutes.

An alternative analytical method for nitrogen dioxide (NO2) in atmosphere was developed. The collection of NO2 is performed by a Sep-Pack C-18 cartridge impregnated with 11% (v/v) of triethanolamine plus 3,6 % (v/v) of ethylene glycol plus 25 % (v/v) of acetone combined solution. When the impregnating is used, NO2 is collected with good repeatibility (CV = 3,3 %). The NO2 absorbed in the sampler was stripped from the sorbent with a methanol 5% (v/v) aqueous solution and was determined by colorimetry as nitrite by using the Griess-Saltzman reagent. The detection limit of 1,4 ppb for 60 min sampling at 0,5 L min-1 flow rate was obtained. Preparation and conditioning procedures for TEA-C-18 cartridge, sampling flow rate, absorption capacity and interference of other species are discussed.

OBJECTIVE: Clinical and surgical outcome of patients with subarachnoid hemorrhage (SAH) due to ruptured aneurysm were assessed in comparison to pre-operative data and risk factors such as previous medical history, clinical presenting condition, CT findings and site of bleeding. METHODS: We evaluated 100 consecutive patients with aneurysmal SAH. Gender, color, history of hypertension, smoking habit, site and size of aneurysm, admittance and before surgery Hunt Hess scale, need for cerebro-spinal fluid shunt, presence of complications during the surgical procedure, Glasgow Outcome Scale, presence of vasospasm and of rebleeding were assessed and these data matched to outcome. For statistical analysis, we applied the chi-squared test or Fisher's test using the pondered kappa coeficient. Kruskal-Wallis test was used for comparison of continue variables. Tendency of proportion was analyzed through Cochran-Armitage test. Significance level adopted was 5%. RESULTS: Patients studied were mainly white, female, without previous history of hypertension and non-smokers. Upon hospital admittance, grade 2 of Hunt-Hess scale was most frequently observed (34%), while grade 3 of Fisher scale was the most prevalent. Single aneurysms were most frequent at anterior circulation, between 12 and 24 mm. The most frequent Glasgow Outcome Scale observed was 5 (60%). Hunt Hess upon the moment of surgery and presence of complications during surgical procedure showed positive correlation with clinical outcome (p=0.00002 and p=0.001, respectively). Other variables were not significantly correlated to prognosis. Tendency of proportion was observed between Hunt-Hess scale and Fisher scale. CONCLUSION: Among variables such as epidemiological data, previous medical history and presenting conditions of patients with ruptured aneurysms, the Hunt-Hess scale upon the moment of surgery and the presence of surgical adversities are statistically related to degree of disability.

In this work, oxidant scrubbers were evaluated for their ability to prevent sampling losses of dimethylsulfide caused by reactions with nitrogen dioxide. Various compounds and mixtures were used in the preparation of the oxidant scrubbers. An automatic flow analysis device was used to compare scrubbing efficiency for nitrogen dioxide. Among the scrubbers tested, the best were shown to be the one made with filter paper or glass wool coated with iron (II) sulfate, sulfuric acid and pyrogallic acid, and the one made from with paper coated with triethanolamine. The results obtained under laboratory conditions, using dimethylsulfide standard gas, and in field experiments confirmed that these scrubbers are suitable for the prevention of oxidation during sampling.

Abstract Background Cow's milk is the most common food allergen in infants and the diagnosis of cow's milk allergy is difficult, even with the use of several diagnostic tests. Therefore, elimination diets and challenge tests are essential for the diagnosis and treatment of this disorder. The aim of this study is to report the clinical presentation and nutritional status of children evaluated by pediatric gastroenterologists for the assessment of symptoms suggestive of cow's milk allergy. Methods An observational cross-sectional study was performed among 9,478 patients evaluated by 30 pediatric gastroenterologists for 40 days in 5 different geographical regions in Brazil. Clinical data were collected from patients with symptoms suggestive of cow's milk allergy. The nutritional status of infants (age ≤ 24 months) seen for the first time was evaluated according to z-scores for weight-for-age, weight-for-height, and height-for-age. Epi-Info (CDC-NCHS, 2000) software was used to calculate z-scores. Results The prevalence of suspected cow's milk allergy in the study population was 5.4% (513/9,478), and the incidence was 2.2% (211/9,478). Among 159 infants seen at first evaluation, 15.1% presented with a low weight-for-age z score (< -2.0 standard deviation - SD), 8.7% with a low weight-for-height z score (< -2.0 SD), and 23.9% with a low height-for-age z score (< -2.0 SD). Conclusion The high prevalence of nutritional deficits among infants with symptoms suggestive of cow's milk allergy indicates that effective elimination diets should be prescribed to control allergy symptoms and to prevent or treat malnutrition.

After serving a prison sentence, sex offenders embark on a community reintegration process, where they usually face several barriers that might negatively affect their successful return to society. Results from semi-structured interviews conducted with sex offenders revealed that participants perceive the economic difficulties, the nature of the crime, and the stigma as the main social factors hindering their community reintegration process. Additionally, prison and parole support are described as insufficient, lacking focus on the specific needs of the participants to act as an effective help. On the contrary, housing and affective relationships were not perceived as major obstacles to a successful reintegration. These findings highlight the need for policies that promote economic opportunities, reduce stigma, and enhance both prison-based and post-release support to facilitate a more effective reintegration process., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Background: Graduate students face higher depression rates worldwide, which were further exacerbated during the COVID-19 pandemic. This study employed a machine learning approach to predict depressive symptoms using academic-related stressors., Methods: We surveyed students across four graduate programs at a Federal University in Brazil between October 15, 2021, and March 26, 2022, when most activities were restricted to taking place online due to the pandemic. Through an online self-reported screening, participants rated ten academic stressors and completed the Patient Health Questionnaire (PHQ-9). Machine learning analysis tested whether the stressors would predict depressive symptoms. Gender, age, and race and ethnicity were used as covariates in the predictive model., Results: Participants (n=172), 67.4 % women, mean age: 28.0 (SD: 4.53) fully completed the online questionnaires. The machine learning approach, employing an epsilon-insensitive support vector regression (Ɛ-SVR) with a k-fold (k=5) cross-validation strategy, effectively predicted depressive symptoms (r=0.51; R 2 =0.26; NMSE=0.79; all p=0.001). Among the academic stressors, those that made the greatest contribution to the predictive model were "fear and worry about academic performance", "financial difficulties", "fear and worry about academic progress and plans", and "fear and worry about academic deadlines"., Conclusions: This study highlights the vulnerability of graduate students to depressive symptoms caused by academic-related stressors during the COVID-19 pandemic through an artificial intelligence methodology. These findings have the potential to guide policy development to create intervention programs and public health initiatives targeted towards graduate students., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Aggregation of alpha-synuclein (aSyn) occurs in presynaptic neurons and constitutes a key factor for the progression of Parkinson's disease, emphasising the urgency of early detection to support effective treatment. Unfortunately, a reliable, sensitive and cost-effective diagnostic tool has so far been lacking. Thus, this work presents a novel biosensor for detecting aSyn using plastic antibodies coupled to electrochemical detection. This biosensor was designed for portability and compatibility with point-of-care devices and exploits the electropolymerization of methylene blue (MB) together with aSyn on the carbon working electrode of screen-printed electrodes (SPEs). By electrochemical impedance spectroscopy (EIS) measurements, the sensor showed exceptional analytical performance in detecting aSyn monomers in human CSF samples. It showed a linear trend of response from 1 fM to 10 pM with an impressively low limit of detection of 69 aM. Selectivity tests confirmed the predominant response to aSyn monomers, a less intense response to oligomers and insensitivity to fibrils. Overall, this plastic antibody-based electrochemical sensor represents a significant breakthrough as it is the first of its kind to accurately, sensitively and selectively detect aSyn monomers with a partial response to oligomers. Its simplicity and reproducibility promise to contribute to the early and effective diagnosis of Parkinson's disease., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The intestinal microbiota is widely recognized as an integral factor in host health, metabolism, and immunity. In this study, the impact of dietary fiber sources on the intestinal microbiota and the production of short-chain fatty acids (SCFAs) was evaluated in Lohmann White laying hens. The hens were divided into four treatment groups: a control diet without fiber, a diet with wheat bran (mixed fibers), a diet with insoluble fiber (cellulose), and a diet with soluble fiber (pectin), with six replicates of four hens each. Cecal content from 24 hens was analyzed using 16 S rRNA sequencing, while SCFA concentrations were measured in blood serum. Alpha diversity analysis revealed significant variations in microbial richness and diversity among treatments, with higher species richness observed in hens fed wheat bran and cellulose, as indicated by Shannon indices. Principal Coordinates Analysis (PCoA) showed significant differences in microbial composition between the control group and the fiber-supplemented groups. The predominant phyla were Bacteroidetes, Campilobacterota, Firmicutes, and Spirochaetota, with a notable increase in Bacteroidetes in fiber-supplemented groups. Regarding SCFAs, fiber inclusion increased acetic and propionic acid concentrations compared to the control group. Diets with mixed fibers (wheat bran) resulted in the highest acetic acid levels, while propionic acid was most abundant in hens fed soluble fiber (pectin). These findings demonstrate that dietary fiber inclusion to laying hens enhances microbial diversity, stimulates SCFA production, and contributes to host metabolism and health., Competing Interests: Declarations. Ethics approval and consent to participate: The procedures adopted were approved by the ethics committee for the use of animals of the Federal University of Minas Gerais (CEUA/UFMG– nº. 220/2021). Consent to participate: Not applicable. Consent for publication: All authors provided consent for the publication of this manuscript. Competing interests: The authors declare no competing interests., (© 2024. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

Inherited retinal dystrophies/degenerations (IRDs) are the leading cause of visual impairment and incurable familial blindness in the Western world. Given the clinical and genetic heterogeneity, establishing a molecular diagnosis is especially relevant. The aim of this study was to perform the first nationwide survey to understand the prevalence and current management of IRDs in Portugal. A response was obtained from 26 healthcare providers (HCP) (76.5% response rate). Only 4 respondents reported not managing IRD patients. Most HCPs (68.1%) reported managing up to 100 patients, while three currently manage between 501 and 1000 patients. Based on the Portuguese population, an estimated IRD prevalence of 0.031%, i.e., about 1 in 3000 individuals, was calculated. In most HCPs (86.3%), most patients are adults, and non-syndromic retinitis pigmentosa is the most frequent diagnosis. Only 4 HCPs currently use the national, web-based IRD registry (IRD-PT). However, all but one respondent expressed interest in participating in such a registry. Genetic testing is available in 54.5%, with 58.3% HCPs reporting solved rates between 61-80%, but 4 to 9 months to get a genetic test result in 83.4% of cases. Based on this survey, the prevalence of biallelic RPE65-associated disease in Portugal is 0.00031%, i.e., approximately 1:300,000 individuals. Data from this study provide vital background information on national differences in the diagnosis and management of IRD patients. Nationwide implementation of the IRD-PT registry should be encouraged and supported to provide population-based reference data and to identify patients eligible for current and future therapies., (© 2024. The Author(s).)

Human parainfluenza virus type 3 (hPIV3) is a respiratory pathogen that can cause severe disease in older people and infants. Currently, vaccines against hPIV3 are in clinical trials but none have been approved yet. The haemagglutinin-neuraminidase (HN) and fusion (F) surface glycoproteins of hPIV3 are major antigenic determinants. Here we describe naturally occurring potently neutralizing human antibodies directed against both surface glycoproteins of hPIV3. We isolated seven neutralizing HN-reactive antibodies and a pre-fusion conformation F-reactive antibody from human memory B cells. One HN-binding monoclonal antibody (mAb), designated PIV3-23, exhibited functional attributes including haemagglutination and neuraminidase inhibition. We also delineated the structural basis of neutralization for two HN and one F mAbs. MAbs that neutralized hPIV3 in vitro protected against infection and disease in vivo in a cotton rat model of hPIV3 infection, suggesting correlates of protection for hPIV3 and the potential clinical utility of these mAbs., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Introduction: Recently, several studies on the 7 vs. 6 "empty goal" (EG) in handball have produced different and even contradictory results. The aim of the present study was to investigate the behavior of teams and players in the 7 vs. 6 EG attack in the European (Euro) and World Championships (WCh) between 2020 and 2023 and characterize the coaches' perceptions., Methods: A mixed-methods approach was used, consisting of the following: (i) an observational methodology and instrument developed and validated to collect observational data on player and team behavior; and (ii) a developed and validated questionnaire to coaches on their perceptions of the 7 vs. 6 game. Observational data were collected during the Euro 2020 and 2022 games ( n  = 62) and the WCh 2021 and 2023 games ( n  = 70). A total of 132 games and 391 situations of 7 vs. 6 attacking sequences were observed. In total, 156 coaches participated (146 men), with a mean age 42.33 ± 11.87 years, 19 nationalities, and with 12.77 ± 9.45 years of experience., Results and Discussion: The choice of 7 vs. 6 offensive play was mostly made in the second half (>73%). The effectiveness of 7 vs. 6 offensive sequences was higher in the top six teams than in the team's ranked 7th to 12th (Euro 2020 51.6%-50.0%; WCh 2021 52.0%-50.0%; Euro 2022 53.1%-41.7%; WCh 2023 50.0%-43.8%). Some patterns of association were found ( p  < 0.05 and with values >±1.96): (i) scoring a goal with a breakthrough shot was significantly associated with the effectiveness of the 7 vs. 6 attack (Euro 2020 2.61; WCh 2021 2.87; Euro 2022 2.68; WCh 2023 2.32); (ii) teams in the top six significantly used 7 vs. 6 when they were winning (Euro 2020 2.17; WCh 2021 3.52; Euro 2022 5.88; WCh 2023 2.54); and (iii) teams in the bottom six used it when they were losing by at least four goals (Euro 2020 7.56; Euro 2022 6.64; WCh 2023 4.37) or when they were winning by four goals or more (WCh 2021 2.58). Coaches that agree with the possibility of playing 7 vs. 6 (74.4%), rarely or never do so (55.6%) because it brings little or no advantage (52.6%). The results of the analysis confirmed the perception of the coaches, the low use of 7 vs. 6, the low advantage associated with it, and the influence of the result and the moment of the game on its use., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2024 Prudente, Cardoso, Rodrigues, Mendes, Fernando, Lopes, Trejo-Silva and Sousa.)

This study aimed to investigate the effects of 8 h of cold stress (18 °C) every day in broiler chicks during the first 7 days of rearing on crop filling analysis, yolk sac consumption, digestive and immune organs weights, and physiological metabolism at seven days and performance between 1 and 35 days. Cobb500 male broiler chickens (n = 274) were randomly assigned to two treatments. The treatments consisted of varying environmental temperatures during the first week post-housing. Chicks were reared at a thermoneutral temperature (32 °C) or under cold stress (18 °C) for 8 h/day during the first week, and both groups were subsequently reared at a thermoneutral temperature for 8-35 days. The thermoneutral group reached 90% full crop after 48 h of housing (P < 0.05), while the cold-stressed group had more empty crops at 2 h and 48 h after housing (P < 0.05). The chick cloacal temperature was not affected by the treatments (P > 0.05). Additionally, the treatment did not affect serum amylase and corticosterone levels, feed intake, body weight gain, or feed conversion ratio (P > 0.05, while the cold-stressed group had elevated heterophil/lymphocyte count at day 7 (P < 0.05). The thermoneutral group showed higher viability (%) at 7 and 35 days and a higher production factor at 35 days (P < 0.05). Broiler chickens under cyclic cold stress experienced decreased yolk sac absorption during the first week and increased feed intake and feed conversion ratio after 35 days of rearing. Viability was also lower in the cold-stressed group. An appropriate strategy to minimize these adverse effects is to rear the chicks in a thermoneutral environment during the first week., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Intervention in Palliative Care aims to provide physical, psychosocial, and spiritual relief for patients and family members. Brief interventions with a psycho-existential approach have shown positive responses; however, cultural adaptations are needed. This pilot study aimed to develop the Meaning of Life Therapy (MLT), a novel psycho-existential intervention, rooted in the Dignity Therapy, Life Review, and Meaning-Centered Psychotherapy. MLT was culturally adapted to the Portuguese context to include questions about forgiveness, apology, reconciliation, farewell, and a legacy document, i.e., the Life Letter . Nine PC cancer patients answered a 14-question MLT protocol, intended to help patients find purpose and meaning in life. Eight themes emerged: Family, Preservation of Identity, Life Retrospective, Clinical Situation, Achievements, Socio-Professional Valorization, Forgiveness/Apology/Reconciliation, and Saying Goodbye. MLT has proved its ability to respond to the psycho-existential needs of PC patients. Further studies should be conducted to gain extensive knowledge of the effectiveness of culturally responsive interventions., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Objective: Conduct a systematic review concerning the literature that reflects whether the callous and unemotional traits present in childhood and/or adolescence are precursors in the development of female psychopathy in adulthood., Materials and Methods: A systematic review involved consulting three databases-EBSCO, the Web of Science, and PubMed-for peer-reviewed and quantitative studies within the period 2000-2023. Nine articles with quality of three and above were included., Results: The presence of callous and unemotional traits designates a group of youth that show characteristics associated with psychopathy, specifically when predicting a more severe and chronic pattern of antisocial behaviour. Children with high rates of callous and unemotional traits, who show symptoms of attention deficit hyperactivity disorder in combination with severe conduct problems, are most likely to show features associated with psychopathy. The multidimensional psychopathy construct is considered a better predictor of future and stable antisocial behaviour than the callous and unemotional traits alone model., Conclusions: According to the studies selected, the callous and unemotional traits in childhood seem to be precursors of female psychopathy in adulthood, but only because of the way they seem to enhance conduct problems, disruptive behaviour disorders, and, as a possible outcome, delinquency and antisocial traits, which may be precursors of future psychopathy.

This work presents an innovative ultra-sensitive biosensor having the Spike protein on carbon-based screen-printed electrodes (SPEs), for monitoring in point-of-care antibodies against SARS-CoV-2, a very important tool for epidemiological monitoring of COVID-19 infection and establishing vaccination schemes. In an innovative and simple approach, a highly conductive support is combined with the direct adsorption of Spike protein to enable an extensive antibody capture. The high conductivity was ensured by using carboxylated carbon nanotubes on the carbon electrode, by means of a simple and quick approach, which also increased the surface area. These were then modified with EDC/NHS chemistry to produce an amine layer and undergo Spike protein adsorption, to generate a stable layer capable of capturing the antibodies against SARS-CoV-2 in serum with great sensitivity. Electrochemical impedance spectroscopy was used to evaluate the analytical performance of this biosensor in serum. It displayed a linear response between 1.0 ​pg/mL and 10 ​ng/mL, with a detection limit of ∼0.7 ​pg/mL. The analysis of human positive sera containing antibody in a wide range of concentrations yielded accurate data, correlating well with the reference method. It also offered the unique ability of discriminating antibody concentrations in sera below 2.3 ​μg/mL, the lowest value detected by the commercial method. In addition, a proof-of-concept study was performed by labelling anti-IgG antibodies with quantum dots to explore a new electrochemical readout based on the signal generated upon binding to the anti-S protein antibodies recognised on the surface of the biosensor. Overall, the alternative serologic assay presented is a promising tool for assessing protective immunity to SARS-CoV-2 and a potential guide for revaccination., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

The dynamic of changes in the rules in team games materialize from research and debate between experts and coaches before being implemented by the International Federations. In Handball, the last changes occurred in 2016, and one of them was to substitute the goalkeeper with an additional field player allowing teams to play "empty goal" while using the additional field player. This study aimed to analyze and characterize the use of the 7 vs. 6 strategical-tactical option for the attack in the 2020 Men's European Championship. We also analyzed whether the game time and partial score influenced the use of 7 vs. 6 and its efficiency. Observational methodology and a mixed ad hoc instrument combining field format and category systems validated by experts were used. Data were taken from 28 matches involving teams in the first 12 positions in the 2020 Men's European Championship. The total number of offensive sequences in an organized attack method in 7 vs. 6 with an empty goal ( n  = 123) were analyzed. Sequential analysis techniques with lags, prospective and retrospective, and polar coordinate analysis were used. The results showed that (a) these solutions had poor efficiency, except for the Portuguese National Team; (b) teams using the 7 vs. 6 tactic option had no negative consequences or increased risk with the opponent response; (c) partial score influenced the decision to use 7 vs. 6 strategic option, and (d) game time was associated with 7 vs. 6 play options and detected patterns., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Prudente, Cardoso, Rodrigues, Mendes, Fernando, Lopes and Sousa.)

The dentinogenic ghost cell tumour (DGCT) is a rare benign neoplasm, which histologically presents itself as an aberrant keratinisation of the epithelium, ghost cells and dentinoid material. Depending on its location there are two different types of DGCT, central or peripheral, with different clinical characteristics. By 2019, there were only 57 cases of DGCT published: 39 of the central type and 18 of the peripheral type.In this clinical case, the authors describe the case of a 78-year-old man with a painless and slow growing mandibular lump. The diagnosis of peripheral DGCT was made by incisional biopsy and the treatment consisted of radical excision with upper marginal mandibulectomy.The aim of the article is to report a clinical case of a rare pathology and, consequently, to help diagnose and better understand its biological behaviour., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Limited 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

Introduction: To date, no valid outcome measure has been developed in European Portuguese (EP) to evaluate the Parkinsons' Disease (PD) patients' (PwP) reports regarding their swallowing disturbances., Objectives: The aim of this study was to translate and cross-culturally adapt the Swallowing Disturbance Questionnaire (SDQ) and the Sialorrhea Clinical Scale for PD (SCS-PD) into EP and to determine its clinimetric properties in PwP., Materials and Methods: The original English SDQ and SCS-PD versions were cross-culturally adapted following recommendations established in international guidelines. The validation process involved 75 PwP and 65 healthy sex- and age-matched participants., Results: The EP versions of the SDQ and SCS-PD are equivalent to the original versions (content, depth, and scoring). Statistical analyses for the SDQ tool revealed good feasibility (missing data <5%), acceptability (no floor or ceiling effects), excellent internal consistency (Cronbach´s α = 0.95), good construct validity (78.5% revealed large to moderate loadings), moderate convergent validity ( r  = 0.60), good divergent validity ( r  = 0.40), good known-groups validity ( p -value < .05) and a fair sensitivity and specificity (AUC = 0.700). Statistical analyses for the SCS-PD tool shows good feasibility, reasonable acceptability (floor effect), good internal consistency (Cronbach´s α = 0.85), good construct validity (85.7% showed between large to moderate loadings), good convergent validity ( r  = 0.78), good divergent validity ( r  = 0.39), good known groups validity ( p -value < .05) and a fair sensitivity and specificity (AUC = 0.704)., Conclusions: The EP versions of the SDQ and SCS-PD maintained the characteristics of the original versions and therefore consistent tools to be used in PwP.

In the past few years, there has been an increasing neuroscientific interest in understanding the function of mammalian chromodomains helicase DNA-binding (CHD) proteins due to their association with severe developmental syndromes. Mammalian CHDs include nine members (CHD1 to CHD9), grouped into subfamilies according to the presence of specific functional domains, generally highly conserved in evolutionary terms. Mutations affecting these domains hold great potential to disrupt protein function, leading to meaningful pathogenic scenarios, such as embryonic defects incompatible with life. Here, we analysed the evolution of CHD proteins by performing a comparative study of the functional domains of CHD proteins between orthologous and paralogous protein sequences. Our findings show that the highest degree of inter-species conservation was observed at Group II (CHD3, CHD4, and CHD5) and that most of the pathological variations documented in humans involve amino acid residues that are conserved not only between species but also between paralogs. The parallel analysis of both orthologous and paralogous proteins, in cases where gene duplications have occurred, provided extra information showing patterns of flexibility as well as interchangeability between amino acid positions. This added complexity needs to be considered when the impact of novel mutations is assessed in terms of evolutionary conservation.

The appearance and quick spread of the new severe acute respiratory syndrome coronavirus disease, COVID-19, brought major societal challenges. Importantly, suitable medical diagnosis procedures and smooth clinical management of the disease are an emergent need, which must be anchored on novel diagnostic methods and devices. Novel molecular diagnostic tools relying on nucleic acid amplification testing have emerged globally and are the current gold standard in COVID-19 diagnosis. However, the need for widespread testing methodologies for fast, effective testing in multiple epidemiological scenarios remains a crucial step in the fight against the COVID-19 pandemic. Biosensors have previously shown the potential for cost-effective and accessible diagnostics, finding applications in settings where conventional, laboratorial techniques may not be readily employed. Paper- and cellulose-based biosensors can be particularly relevant in pandemic times, for the renewability, possibility of mass production with sustainable methodologies, and safe environmental disposal. In this review, paper-based devices and platforms targeting SARS-CoV-2 are showcased and discussed, as a means to achieve quick and low-cost PoC diagnosis, including detection methodologies for viral genomic material, viral antigen detection, and serological antibody testing. Devices targeting inflammatory markers relevant for COVID-19 are also discussed, as fast, reliable bedside diagnostic tools for patient treatment and follow-up., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

Understanding the role of common polymorphisms in modulating the clinical phenotype when they co-occur with a disease-causing lesion is of critical importance in medical genetics. We explored the impact of apparently neutral common polymorphisms, using the gene encoding the urea cycle enzyme, ornithine transcarbamylase (OTC), as a model system. Distinct combinations of genetic backgrounds embracing two missense polymorphisms were created in cis with the pathogenic p.Arg40His replacement. In vitro enzymatic assays revealed that the polymorphic variants were able to modulate OTC activity both in the presence or absence of the pathogenic lesion. First, we found that the combination of the minor alleles of polymorphisms p.Lys46Arg and p.Gln270Arg significantly enhanced enzymatic activity in the wild-type protein. Second, enzymatic assays revealed that the minor allele of the p.Gln270Arg polymorphism was capable of ameliorating OTC activity when combined in cis with the pathogenic p.Arg40His replacement. Structural analysis predicted that the minor allele of the p.Gln270Arg polymorphism would serve to stabilize the OTC wild-type protein, thereby corroborating the results of the experimental assays. Our findings demonstrate the potential importance of cis-interactions between common polymorphic variants and pathogenic missense mutations and illustrate how standing genetic variation can modulate protein function., (© 2021 The Authors. Human Mutation Published by Wiley Periodicals LLC.)

As part of the biomimetic enzyme field, nanomaterial-based artificial enzymes, or nanozymes, have been recognized as highly stable and low-cost alternatives to their natural counterparts. The discovery of enzyme-like activities in nanomaterials triggered a broad range of designs with various composition, size, and shape. An overview of the properties of nanozymes is given, including some examples of enzyme mimics for multiple biosensing approaches. The limitations of nanozymes regarding lack of selectivity and low catalytic efficiency may be surpassed by their easy surface modification, and it is possible to tune specific properties. From this perspective, molecularly imprinted polymers have been successfully combined with nanozymes as biomimetic receptors conferring selectivity and improving catalytic performance. Compelling works on constructing imprinted polymer layers on nanozymes to achieve enhanced catalytic efficiency and selective recognition, requisites for broad implementation in biosensing devices, are reviewed. Multimodal biomimetic enzyme-like biosensing platforms can offer additional advantages concerning responsiveness to different microenvironments and external stimuli. Ultimately, progress in biomimetic imprinted nanozymes may open new horizons in a wide range of biosensing applications.

Testicular germ cell tumours (TGCTs) are heterogeneous neoplasms mostly affecting young-adult men. Despite high survival rates, some patients with disseminated disease acquire cisplatin resistance, entailing the need for less toxic therapies. Epigenetic alterations constitute an important feature of TGCTs, which are also implicated in resistance mechanism(s). These alterations might be used as potential targets to design epigenetic drugs. To date, several compounds have been explored and evaluated regarding therapeutic efficacy, making use of pre-clinical studies with in vitro and in vivo models, and some have already been explored in clinical trials. This review summarizes the several epigenetic mechanisms at play in these neoplasms, the current challenges in the field of TGCTs and critically reviews available data on 'epidrugs' in those tumours.

Less toxic treatment strategies for testicular germ cell tumor (TGCT) patients are needed, as overtreatment is a concern due to the long-term side effects of platin-based chemotherapy. Although clinical benefit from classical hypomethylating agents has to date been limited, TGCTs show an abnormal DNA methylome indicating the potential of treating TGCTs with hypomethylating drugs. We tested, for the first time in TGCT cell lines, a new synthetic flavonoid compound (MLo1302) from the 3-nitroflavanone family of DNA methyltransferase (DNMT) inhibitors. We show that MLo1302 reduces cell viability (including of cisplatin resistant cell line NCCIT-R), with IC 50 s (inhibitory concentration 50) within the nanomolar range for NCCIT and NTERA-2 cells, and proved its cytotoxic effect. Exposure to MLo1302 reduced DNMT protein expression, similar to decitabine, and showed a partial effect in cell differentiation, reducing protein expression of pluripotency markers. RT 2 profiler expression array indicated several dysregulated targets, related to activation of apoptosis, differentiation, and cell cycle arrest. We validated these data by showing increased apoptosis, increased protein expression of cleaved caspase 8 and activated caspase 2, and reduced proliferation (BrdU assay), with increase in CDKN1A and decrease in MIB-1 expression. Therefore, synthetic drugs designed to target DNA methylation in cells may uncover effective treatments for TGCT patients.

SARS-CoV-2-specific antibodies, particularly those preventing viral spike receptor binding domain (RBD) interaction with host angiotensin-converting enzyme 2 (ACE2) receptor, can neutralize the virus. It is, however, unknown which features of the serological response may affect clinical outcomes of COVID-19 patients. We analyzed 983 longitudinal plasma samples from 79 hospitalized COVID-19 patients and 175 SARS-CoV-2-infected outpatients and asymptomatic individuals. Within this cohort, 25 patients died of their illness. Higher ratios of IgG antibodies targeting S1 or RBD domains of spike compared to nucleocapsid antigen were seen in outpatients who had mild illness versus severely ill patients. Plasma antibody increases correlated with decreases in viral RNAemia, but antibody responses in acute illness were insufficient to predict inpatient outcomes. Pseudovirus neutralization assays and a scalable ELISA measuring antibodies blocking RBD-ACE2 interaction were well correlated with patient IgG titers to RBD. Outpatient and asymptomatic individuals' SARS-CoV-2 antibodies, including IgG, progressively decreased during observation up to five months post-infection., (Copyright © 2020, American Association for the Advancement of Science.)

B cells are critical for the production of antibodies and protective immunity to viruses. Here we show that patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) who develop coronavirus disease 2019 (COVID-19) display early recruitment of B cells expressing a limited subset of IGHV genes, progressing to a highly polyclonal response of B cells with broader IGHV gene usage and extensive class switching to IgG and IgA subclasses with limited somatic hypermutation in the initial weeks of infection. We identify convergence of antibody sequences across SARS-CoV-2-infected patients, highlighting stereotyped naive responses to this virus. Notably, sequence-based detection in COVID-19 patients of convergent B cell clonotypes previously reported in SARS-CoV infection predicts the presence of SARS-CoV/SARS-CoV-2 cross-reactive antibody titers specific for the receptor-binding domain. These findings offer molecular insights into shared features of human B cell responses to SARS-CoV-2 and SARS-CoV., Competing Interests: Declaration of Interests A.T.S. is a scientific founder of Immunai and receives research funding from Arsenal Biosciences not related to this study. M.H. is an employee of ATUM. S.D.B. has consulted for Regeneron, Sanofi, and Novartis on topics unrelated to this study. S.D.B., K.R., P.S.K., and A.E.P. have filed provisional patent applications related to serological tests for SARS-CoV-2 antibodies. K.C.N. reports grants from National Institute of Allergy and Infectious Diseases (NIAID), Food Allergy Research & Education (FARE), and End Allergies Together (EAT); National Heart, Lung, and Blood Institute (NHLBI), and National Institute of Environmental Health Sciences (NIEHS), and is the director of FARE and World Allergy Organization (WAO) Center of Excellence at Stanford; advisor at Cour Pharma; co-founder of Before Brands, Alladapt, Latitude, and IgGenix; National Scientific Committee member at Immune Tolerance Network (ITN) and National Institutes of Health (NIH); a recipient of a Research Sponsorship from Nestle; Consultant and Advisory Board Member at Before Brands, Alladapt, Iggenix, NHLBI, and Probio; Data and Safety Monitoring Board member at NHLBI; and has US patents for basophil testing, multifood immunotherapy and prevention, monoclonal antibodies from plasmablasts, and devices for diagnostics. The remaining authors declare that they have no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

SARS-CoV-2-specific antibodies, particularly those preventing viral spike receptor binding domain (RBD) interaction with host angiotensin-converting enzyme 2 (ACE2) receptor, could offer protective immunity, and may affect clinical outcomes of COVID-19 patients. We analyzed 625 serial plasma samples from 40 hospitalized COVID-19 patients and 170 SARS-CoV-2-infected outpatients and asymptomatic individuals. Severely ill patients developed significantly higher SARS-CoV-2-specific antibody responses than outpatients and asymptomatic individuals. The development of plasma antibodies was correlated with decreases in viral RNAemia, consistent with potential humoral immune clearance of virus. Using a novel competition ELISA, we detected antibodies blocking RBD-ACE2 interactions in 68% of inpatients and 40% of outpatients tested. Cross-reactive antibodies recognizing SARS-CoV RBD were found almost exclusively in hospitalized patients. Outpatient and asymptomatic individuals' serological responses to SARS-CoV-2 decreased within 2 months, suggesting that humoral protection may be short-lived., Competing Interests: Competing interests: The authors declare no competing interests.

The gene encoding the cytosolic β-glucosidase GBA3 shows pseudogenization due to a truncated allele (rs358231) that is polymorphic in humans. Since this enzyme is involved in the transformation of many plant β-glycosides, this particular case of gene loss may have been influenced by dietary adaptations during evolution. In humans, apart from the inactivating allele, we found that GBA3 accumulated additional damaging mutations, implying an extensive GBA3 loss. The allelic distribution of loss-of-function alleles revealed significant differences between human populations which can be partially related with their staple diet. The analysis of mammalian orthologs disclosed that GBA3 underwent at least nine pseudogenization events. Most events of pseudogenization occurred in carnivorous lineages, suggesting a possible link to a β-glycoside poor diet. However, GBA3 was also lost in omnivorous and herbivorous species, hinting that the physiological role of GBA3 is not fully understood and other unknown causes may underlie GBA3 pseudogenization. Such possibility relies upon a putative role in sialic acid biology, where GBA3 participates in a cellular network involving NEU2 and CMAH. Overall, our data shows that the recurrent loss of GBA3 in mammals is likely to represent an evolutionary endpoint of the relaxation of selective constraints triggered by diet-related factors.

Rationale: Prepulse inhibition of the startle reflex (PPI) is disrupted in several psychiatric disorders including schizophrenia. Understanding PPI pharmacology may help elucidate the pathophysiology of these disorders and lead to better treatments. Given the advantages of multi-target approaches for complex mental illnesses treatment, we have investigated the interaction between receptors known to modulate PPI (5-HT 1A and 5-HT 2A ) and the neuromodulatory endocannabinoid system., Objectives: To investigate serotonin and cannabinoid receptor (CBR) co-modulation in a model of PPI disruption relevant to schizophrenia METHODS: Male Swiss mice were pretreated with WIN 55,212-2 (CBR agonist), rimonabant (CB1R inverse agonist), 8-OH-DPAT (5-HT 1A/7 agonist), and volinanserin (5-HT 2A antagonist) or with a combination of a cannabinoid and a serotonergic drug. PPI disruption was induced by acute administration of MK-801., Results: WIN 55,212-2 and rimonabant did not change PPI nor block MK-801-induced deficits. 8-OH-DPAT increased PPI in control mice and, in a higher dose, inhibited MK-801-induced impairments. Volinanserin also increased PPI in control and MK-801-treated mice, presenting an inverted U-shaped dose-response curve. Co-administration of either cannabinoid ligand with 8-OH-DPAT did not change PPI; however, the combination of volinanserin with rimonabant increased PPI in both control and MK-801-exposed mice., Conclusions: WIN 55,212-2 and rimonabant had similar effects in PPI. Moreover, serotonin and cannabinoid receptors interact to modulate PPI. While co-modulation of CBR and 5-HT 1A receptors did not change PPI, a beneficial effect of 5-HT 2A and CB1R antagonist combination was detected, possibly mediated through potentiation of 5-HT 2A blockade effects by concomitant CB1R blockade.

This work reports the first electrochemical molecularly imprinted polymer (MIP) sensor for Interleukin-1beta (IL-1β) detection, based on modified commercial screen-printed carbon electrode (SPCE) was successfully demonstrated. For this purpose, the carbon support was modified with a PEDOT/4-aminothiophenol layer prior to the MIP film to enhance sensitivity and signal stability. The MIP layer was constructed on top of this by electropolymerization of Eriochrome black T (EBT) in the presence of IL-1β. The several steps of the biosensor assembly was followed by Raman spectroscopy and electroanalytical techniques. Using electrochemical impedance spectroscopy (EIS), a linear response in the range of 60 pM to 600 nM, with a LOD of 1.5 pM with (S/N = 3) was obtained in neutral PBS. Selectivity tests of the MIP biosensor made in spiked synthetic serum samples as well as against other structurally related (Myoglobin, of similar shape and size) or competing compounds (Immunoglobulin G, also present in the human serum) confirmed the good selectivity of the biosensor. Overall, the biosensor described herein has the potential to provide a simple and quick way for on-site screening of IL-1β, with low sample/reagent consumption and enabling direct serum analysis, which constitutes a valuable alternative to other conventional methods., (Copyright © 2019 Elsevier B.V. All rights reserved.)

In the last decades, observational methodology has been widely used in scientific research in sport, namely in the study of team games like handball, due to its characteristics. Handball, in addition to being a collective, complex, dynamic, and interactive sport, has its own characteristics and demands a performance analysis that takes into account the context and interaction between different factors and variables. The present study analyzes how the different numerical relations in an attack can change the center back's patterns of tactical behavior. Observational methodology and a mixed ad hoc instrument combining field format and category systems appropriately validated by experts were used. Data were taken from 20 matches involving teams classified in the first four places in the 2017 Men's World Championship. These were recorded from TV broadcasts, and the total number of offensive sequences carried out in an organized attack game method ( n = 990) was analyzed. In each of the sequences carried out in an organized attack, the numerical relation in attack or defense was observed and recorded. In addition to this, it was verified whether the attacking team maintained or replaced the goalkeeper by a field player in the attack. Both sequential analysis techniques with lags, prospective, and retrospective, as well as polar coordinate analysis, were used. Results have shown that there are different behavioral patterns of the center back in the three different situations of numerical relation. Another element that stood out was that in numerical equality with the defense and no goalkeeper at the goal, the center back opted for greater security and less risk of loss of the ball., (Copyright © 2019 Prudente, Cardoso, Rodrigues and Sousa.)

Neurodevelopmental disorders (NDDs) represent a growing medical challenge in modern societies. Ever-increasing sophisticated diagnostic tools have been continuously revealing a remarkably complex architecture that embraces genetic mutations of distinct types (chromosomal rearrangements, copy number variants, small indels, and nucleotide substitutions) with distinct frequencies in the population (common, rare, de novo). Such a network of interacting players creates difficulties in establishing rigorous genotype-phenotype correlations. Furthermore, individual lifestyles may also contribute to the severity of the symptoms fueling a large spectrum of gene-environment interactions that have a key role on the relationships between genotypes and phenotypes.Herein, a review of the genetic discoveries related to NDDs is presented with the aim to provide useful general information for the medical community.

Bcl-2 protein is involved in cell apoptosis and is considered an interesting target for anti-cancer therapy. The present study aims to understand the stability and conformational changes of Bcl-2 upon interaction with the inhibitor venetoclax, and to explore other drug-target regions. We combined biophysical and in silico approaches to understand the mechanism of ligand binding to Bcl-2. Thermal shift assay (TSA) and urea electrophoresis showed a significant increase in protein stability upon venetoclax incubation, which is corroborated by molecular docking and molecular dynamics simulations. An 18 °C shift in Bcl-2 melting temperature was observed in the TSA, corresponding to a binding affinity multiple times higher than that of any other reported Bcl-2 inhibitor. This protein-ligand interaction does not implicate alternations in protein conformation, as suggested by SAXS. Additionally, bioinformatics approaches were used to identify deleterious non-synonymous single nucleotide polymorphisms (nsSNPs) of Bcl-2 and their impact on venetoclax binding, suggesting that venetoclax interaction is generally favored against these deleterious nsSNPs. Apart from the BH3 binding groove of Bcl-2, the flexible loop domain (FLD) also plays an important role in regulating the apoptotic process. High-throughput virtual screening (HTVS) identified 5 putative FLD inhibitors from the Zinc database, showing nanomolar affinity toward the FLD of Bcl-2.

Graphene has emerged as a novel material with enhanced electrical and structural properties that can be used for a multitude of applications from super-capacitors to biosensors. In this context, an ultra-sensitive biosensor was developed using a low-cost, simple and mask-free method based on laser-induced graphene technique for electrodes patterning. The graphene was produced on a polyimide substrate, showing a porous multi-layer structure with a resistivity of 102.4 ± 7.3 Ω/square. The biosensor was designed as a 3-electrode system. Auxiliary and working electrodes were made of graphene by laser patterning and the reference electrode was handmade by casting a silver ink. A molecularly-imprinted polymer (MIP) was produced at the working electrode by direct electropolymerization of eriochrome black T (EBT). As proof-of-concept, the MIP film was tailored for chloramphenicol (CAP), a common contaminant in aquaculture. The resulting device was evaluated by cyclic voltammetry and electrochemical impedance spectroscopy readings against a redox standard probe. The limit of detection (LOD) was 0.62 nM and the linear response ranged from 1 nM to 10 mM. These analytical features were better than those produced by assembling the same biorecognition element on commercial graphene- and carbon-based screen-printed electrodes. Overall, the simplicity and quickness of the laser-induced graphene technique, along with the better analytical features obtained with the graphene-based electrodes, shows the potential to become a commercial approach for on-site sensing., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The dopaminergic system plays important roles in neuromodulation, such as motor control, motivation, reward, cognitive function, maternal, and reproductive behaviors. Dopamine is a neurotransmitter, synthesized in both central nervous system and the periphery, that exerts its actions upon binding to G protein-coupled receptors. Dopamine receptors are widely expressed in the body and function in both the peripheral and the central nervous systems. Dopaminergic signaling pathways are crucial to the maintenance of physiological processes and an unbalanced activity may lead to dysfunctions that are related to neurodegenerative diseases. Unveiling the neurobiology and the molecular mechanisms that underlie these illnesses may contribute to the development of new therapies that could promote a better quality of life for patients worldwide. In this review, we summarize the aspects of dopamine as a catecholaminergic neurotransmitter and discuss dopamine signaling pathways elicited through dopamine receptor activation in normal brain function. Furthermore, we describe the potential involvement of these signaling pathways in evoking the onset and progression of some diseases in the nervous system, such as Parkinson's, Schizophrenia, Huntington's, Attention Deficit and Hyperactivity Disorder, and Addiction. A brief description of new dopaminergic drugs recently approved and under development treatments for these ailments is also provided.

Zika virus (ZIKV) recently emerged as a global threat subsequent to its global spread because it induces microencephaly and other brain damages in infants born to infected mothers. Epidemiological monitoring of infection has been hampered by the absence of reliable serological tests capable to distinguish between ZIKV and other Flavivirus infections, in particular Dengue virus (DENV). As both viruses are transmitted by the same mosquito-species, their distributions largely overlap and reliable serological distinction between the viruses is essential. Here we develop a novel biosensor which is based on recombinant forms of ZIKV non-structural protein 1 (NS1) and the domain III of the envelope protein (EDIII). Using electrochemical impedance spectroscopy (EIS) and square wave voltammetry (SWV), we demonstrate that in addition to extremely sensitive detection of ZIKV-specific antibodies in serum and saliva, the biosensor promptly distinguished ZIKV and DENV-specific antibodies. Hence, this novel biosensor allows assessing ZIKV antibodies in blood and saliva and results are unaffected by presence of DENV virus-specific antibodies., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Selective base pairing is the foundation of DNA recognition. Here, we elucidate the molecular and structural details of a FRET-based two-component molecular beacon relying on steady-state fluorescence spectroscopy, small-angle X-ray scattering (SAXS), microscale thermophoresis (MST), and differential electrophoretic mobility. This molecular beacon was designed to detect the most common fusion sequences causing chronic myeloid leukemia, e14a2 and e13a2. The emission spectra indicate that the self-assembly of the different components of the biosensor occurs sequentially, triggered by the fully complementary target. We further assessed the structural alterations leading to the specific fluorescence FRET signature by SAXS, MST, and the differential electrophoretic mobility, where the size range observed is consistent with hybridization and formation of a 1:1:1 complex for the probe in the presence of the complementary target and revelator. These results highlight the importance of different techniques to explore conformational DNA changes in solution and its potential to design and characterize molecular biosensors for genetic disease diagnosis.

Molybdenum and tungsten are taken up by bacteria and archaea as their soluble oxyanions through high affinity transport systems belonging to the ATP-binding cassette (ABC) transporters. The component A (ModA/TupA) of these transporters is the first selection gate from which the cell differentiates between MoO 4 2- , WO 4 2- and other similar oxyanions. We report the biochemical characterization and the crystal structure of the apo-TupA from Desulfovibrio desulfuricans G20, at 1.4 Å resolution. Small Angle X-ray Scattering data suggests that the protein adopts a closed and more stable conformation upon ion binding. The role of the arginine 118 in the selectivity of the oxyanion was also investigated and three mutants were constructed: R118K, R118E and R118Q. Isothermal titration calorimetry clearly shows the relevance of this residue for metal discrimination and oxyanion binding. In this sense, the three variants lost the ability to coordinate molybdate and the R118K mutant keeps an extremely high affinity for tungstate. These results contribute to an understanding of the metal-protein interaction, making it a suitable candidate for a recognition element of a biosensor for tungsten detection.

Sensitive detection of specific antibodies by biosensors has become of major importance for monitoring and controlling epidemics. Here we report a development of a biosensor able to specifically measure antibodies in a drop of unmodified blood serum. Within minutes, the detection system measures presence of antibodies against Plasmodium vivax, a causing agent for malaria. The biosensor consists of a layer of carbon nanotubes (CNTs) which were casted on a carbon working electrode area of a three-electrode system and oxidized. An amine layer was produced next by modifying the surface with EDAC/NHS followed by reaction with a diamine compound. Finally, the protein fragments derived from P. vivax containing well-known antigen sequences were casted on this layer and bound through electrostatic interactions, involving hydrogen and ionic bonding. All these chemical changes occurring at the carbon surface along the biosensor assembly were followed and confirmed by Fourier Transformed Infrared s pectrometry (FTIR) and Raman spectroscopy. The presence of antibodies in serum was detected by monitoring the electrical properties of the layer, making use of cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and square wave voltammetry (SWV), against a standard iron probe. Overall, the charge-transfer resistance decreased after antibody binding, because there was an additional amount of protein bound to the surface. This hindered the access of the iron redox probe to the conductive support at the electrode surface. Electrical changes could be measured at antibody concentration as low as ~6-50pg/L (concentrations in the range of 10-15M) and as high as ~70μg/L. Specific measurement with low background was even possible in undiluted serum. Hence, this novel biosensor allows assessing serum antibody levels in real time and in un-manipulated serum samples on-site where needed., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Introduction: Of all syphilis-related pregnancy outcomes, fetal death is certainly the most common one, being directly related to the availability, accessibility and quality of prenatal care. The present study aimed to analyze the underreporting of fetal and infant deaths and other maternal factors associated with congenital syphilis (CS) death., Methods: This cross-sectional study integrated data of infants that were diagnosed and/or died of CS from the Sistema de Informação de Agravos de Notificação-Sinan (Notifiable Diseases Information System) and the Sistema de Informação de Mortalidade-SIM (Mortality Information System) in Fortaleza, Northeastern Brasil to identify unreported cases of congenital syphilis. We assessed data during the period from 2007 to 2013., Results: The underreporting of CS as a cause of fetal or infant death increased from 41 to 415 cases (90.1%) during 2007-2013. Exactly 3,209 cases of CS were identified in Sinan and 6,578 deaths in SIM. After database linkage, we identified 382 cases that were reported in the SIM and SINAN databases consisting of 309 fetal deaths and 73 infant deaths related to CS. From the children notified at Sinan that born alive, 3.0% (78/2,542) died; Out of these, 39 (50.0%) were early and 25 (32.1%) were late neonatal deaths. The proportion of death by CS increased from 0.62 to 5.8 from 2007 to 2013. At logistic regression, the variable that maintained statistical significance with fetal and infant death outcomes was the presence of CS signs and/or symptoms at birth (OR = 3.20; IC 95% 1.54-6.62; p = 0.002)., Conclusions: Neonatal and Infant deaths following CS-associated live births are underreported in Northeastern Brazil. Data base linkage identified unreported fetal and neonatal deaths due to CS leading to an increased awareness of fetal/infant mortality due to this infection., Competing Interests: The authors have declared that no competing interests exist.

The xanthine oxidase (XO) family comprises molybdenum-dependent enzymes that usually form homodimers (or dimers of heterodimers/trimers) organized in three domains that harbor two [2Fe-2S] clusters, one FAD, and a Mo cofactor. In this work, we crystallized an unusual member of the family, the periplasmic aldehyde oxidoreductase PaoABC from Escherichia coli. This is the first example of an E. coli protein containing a molybdopterin-cytosine-dinucleotide cofactor and is the only heterotrimer of the XO family so far structurally characterized. The crystal structure revealed the presence of an unexpected [4Fe-4S] cluster, anchored to an additional 40 residues subdomain. According to phylogenetic analysis, proteins containing this cluster are widely spread in many bacteria phyla, putatively through repeated gene transfer events. The active site of PaoABC is highly exposed to the surface with no aromatic residues and an arginine (PaoC-R440) making a direct interaction with PaoC-E692, which acts as a base catalyst. In order to understand the importance of R440, kinetic assays were carried out, and the crystal structure of the PaoC-R440H variant was also determined.

Copy number variants (CNVs) are important contributors to the human pathogenic genetic diversity as demonstrated by a number of cases reported in the literature. The high homology between repetitive elements may guide genomic stability which will give rise to CNVs either by non-allelic homologous recombination (NAHR) or non-homologous end joining (NHEJ). Here, we present a short guide based on previously documented cases of disease-associated CNVs in order to provide a general view on the impact of repeated elements on the stability of the genomic sequence and consequently in the origin of the human pathogenic variome.

This work, describes for the first time, a simple biosensing design to yield an ultrasensitive electrochemical biosensor for a cancer biomarker detection, miRNA-155, with linear response down to the attomolar range. MiRNA-155 was selected for being overexpressed in breast cancer. The biosensor was assembled in two stages: (1) the immobilization of the anti-miRNA-155 that was thiol modified on an Au-screen printed electrode (Au-SPE), followed by (2) blocking the areas of non-specific binding with mercaptosuccinic acid. Atomic force microscopy (AFM) and electrochemical techniques including cyclic voltammetry (CV), impedance spectroscopy (EIS) and square wave voltammetry (SWV) confirmed the surface modification of these devices and their ability to hybridize successfully and stably with miRNA-155. The final biosensor provided a sensitive detection of miRNA-155 from 10 aM to 1.0 nM with a low detection limit (LOD) of 5.7 aM in real human serum samples. Good results were obtained in terms of selectivity towards breast cancer antigen CA-15.3 and bovine serum albumin (BSA). Raw fluid extracts from cell-lines of melanoma did not affect the biosensor response (no significant change of the blank), while raw extracts from breast cancer yielded a positive signal against miRNA-155. This simple and sensitive strategy is a promising alternative for simultaneous quantitative analysis of multiple miRNA in physiological fluids for biomedical research and point-of-care (POC) diagnosis., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

In this study, a leaf extract from Schinus terebinthifolius was evaluated for effects on survival, development, and midgut of A. aegypti fourth instar larvae (L4), as well as for toxic effect on Artemia salina. Leaf extract was obtained using 0.15 M NaCl and evaluated for phytochemical composition and lectin activity. Early L4 larvae were incubated with the extract (0.3-1.35%, w/v) for 8 days, in presence or absence of food. Polymeric proanthocyanidins, hydrolysable tannins, heterosid and aglycone flavonoids, cinnamic acid derivatives, traces of steroids, and lectin activity were detected in the extract, which killed the larvae at an LC50 of 0.62% (unfed larvae) and 1.03% (fed larvae). Further, the larvae incubated with the extract reacted by eliminating the gut content. No larvae reached the pupal stage in treatments at concentrations between 0.5% and 1.35%, while in the control (fed larvae), 61.7% of individuals emerged as adults. The extract (1.0%) promoted intense disorganization of larval midgut epithelium, including deformation and hypertrophy of cells, disruption of microvilli, and vacuolization of cytoplasms, affecting digestive, enteroendocrine, regenerative, and proliferating cells. In addition, cells with fragmented DNA were observed. Separation of extract components by solid phase extraction revealed that cinnamic acid derivatives and flavonoids are involved in larvicidal effect of the extract, being the first most efficient in a short time after larvae treatment. The lectin present in the extract was isolated, but did not show deleterious effects on larvae. The extract and cinnamic acid derivatives were toxic to A. salina nauplii, while the flavonoids showed low toxicity. S. terebinthifolius leaf extract caused damage to the midgut of A. aegypti larvae, interfering with survival and development. The larvicidal effect of the extract can be attributed to cinnamic acid derivatives and flavonoids. The data obtained using A. salina indicates that caution should be used when employing this extract as a larvicidal agent.

Enhanced mitochondrial generation of oxidants, including hydrogen peroxide (H2O2), is related to a large number of pathological conditions, including diet-induced obesity and steatohepatosis. Indeed, we have previously shown that high fat diets increase the generation of H2O2 in liver mitochondria energized by activated fatty acids. Here, we further study fatty-acid induced H2O2 release in liver mitochondria, and determine the characteristics that regulate it. We find that this production of H2O2 is independent of mitochondrial inner membrane integrity and insensitive to purine nucleotides. On the other hand, palmitate-induced H2O2 production is strongly enhanced by high fat diets and is pH-sensitive, with a peak at a matrix pH of ~8.5. Using recombinantly expressed human very long chain acyl-CoA dehydrogenase, we are able to demonstrate that palmitate-induced H2O2 release may be ascribed to the activity of this enzyme alone, acting as an oxidase. Our results add to a number of findings indicating that sources outside of the electron transport chain can generate significant, physiopathologically relevant, amounts of oxidants in mitochondria., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

The TupABC system is involved in the cellular uptake of tungsten and belongs to the ABC (ATP binding cassette)-type transporter systems. The TupA component is a periplasmic protein that binds tungstate anions, which are then transported through the membrane by the TupB component using ATP hydrolysis as the energy source (the reaction catalyzed by the ModC component). We report the heterologous expression, purification, determination of affinity binding constants and crystallization of the Desulfovibrio alaskensis G20 TupA. The tupA gene (locus tag Dde&#95;0234) was cloned in the pET46 Enterokinase/Ligation-Independent Cloning (LIC) expression vector, and the construct was used to transform BL21 (DE3) cells. TupA expression and purification were optimized to a final yield of 10 mg of soluble pure protein per liter of culture medium. Native polyacrylamide gel electrophoresis was carried out showing that TupA binds both tungstate and molybdate ions and has no significant interaction with sulfate, phosphate or perchlorate. Quantitative analysis of metal binding by isothermal titration calorimetry was in agreement with these results, but in addition, shows that TupA has higher affinity to tungstate than molybdate. The protein crystallizes in the presence of 30% (w/v) polyethylene glycol 3350 using the hanging-drop vapor diffusion method. The crystals diffract X-rays beyond 1.4 Å resolution and belong to the P21 space group, with cell parameters a = 52.25 Å, b = 42.50 Å, c = 54.71 Å, β = 95.43°. A molecular replacement solution was found, and the structure is currently under refinement.

Molybdenum and tungsten enzymes require specific chaperones for folding and cofactor insertion. PaoD is the chaperone of the periplasmic aldehyde oxidoreductase PaoABC. It is the last gene in the paoABCD operon in Escherichia coli and its presence is crucial for obtaining mature enzyme. PaoD is an unstable, 35 kDa, protein. Our biochemical studies showed that it is a dimer in solution with a tendency to form large aggregates, especially after freezing/thawing cycles. In order to improve stability, PaoD was thawed in the presence of two ionic liquids [C4mim]Cl and [C2OHmim]PF6 and no protein precipitation was observed. This allowed protein concentration and crystallization using polyethylene glycol or ammonium sulfate as precipitating agents. Saturation transfer difference - nuclear magnetic resonance (STD-NMR) experiments have also been performed in order to investigate the effect of the ionic liquids in the stabilization process, showing a clear interaction between the acidic ring protons of the cation and, most likely, negatively charged residues at the protein surface. DLS assays also show a reduction of the overall size of the protein aggregates in presence of ionic liquids. Furthermore, cofactor binding studies on PaoD showed that the protein is able to discriminate between molybdenum and tungsten bound to the molybdenum cofactor, since only a Mo-MPT form of the cofactor remained bound to PaoD.