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In this study, the chemical composition of the essential oil (EO) extracted from Croton blanchetianus Baill leaves was identified, and antimicrobial and antibiofilm activities against Staphylococcus aureus, Staphylococcus epidermidis, and Escherichia coli strains were determined. Moreover, the effects of EO in combination with ampicillin and tetracycline were investigated. Thirty-four components, mainly mono-and sesquiterpenes that represented 94.05 % of the chemical composition, were identified in the EO. The EO showed bacteriostatic and bactericidal activities against all strains tested. Furthermore, the EO showed a synergistic effect with ampicillin and tetracycline. EO significantly inhibited biofilm formation and reduced the number of viable cells in biofilms. The EO may be a promising natural product for preventing bacterial biofilm infections., (© 2022 Wiley-VHCA AG, Zurich, Switzerland.)

The limitations of silica-based sorbents boosted the development of new extraction phases. In this study, boron nitride nanotubes functionalized with octadecyl groups were used for the first time as sorbent for extraction of losartan and valsartan, the most used angiotensin receptor blockers in the clinical practice, from human plasma. The nanotubes were synthesized using the chemical vapor deposition technique, purified by acid treatment, functionalized with octadecylamine in a microwave reactor, and characterized by different techniques. The functionalized nanotubes were packed in solid phase extraction cartridges. Extraction conditions were optimized by means of a 2 3 factorial design with center points. The separation was performed on a biphenyl core-shell (100 × 4.6 mm; 2.6 µm) column, using 0.1 % (v/v) triethylamine solution and methanol (pH 3.2) as mobile phase, at 0.7 mL/min, in gradient elution. The injection volume was 10 µL and fluorescence detection was performed at excitation and emission wavelengths of 250 and 375 nm, respectively. The developed method was validated according to Brazilian Health Regulatory Agency (ANVISA), United States Food and Drug Administration (US FDA) and European Medicines Agency (EMA) guidelines and presented selectivity, precision, accuracy, and linearity in the concentration ranges of 50-1200 ng/mL for losartan and 20-1700 ng/mL for valsartan. Recoveries higher than 80 % were obtained. The method was fit for the quantification of losartan in plasma samples from patients under antihypertensive therapy, being useful in therapeutic drug monitoring, pharmacokinetics and bioequivalence studies., 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 © 2022 Elsevier B.V. All rights reserved.)

Shiga toxin-producing Escherichia coli (STEC) is a foodborne pathogen capable of causing illness in humans. In a previous study, our group showed that a STEC isolate belonging to O22:H8 serotype (strain 154) can interfere with STEC O157:H7 colonization both in vitro and in vivo. Using whole-genome sequencing and genomic comparative, we predicted a subset of genes acquired by O22:H8 strain 154 through horizontal gene transfer that might be responsible for the phenotype previously described by our group. Among them were identified genes related to the pathogenesis of non-LEE (locus of enterocyte effacement) STEC, specific metabolic processes, antibiotic resistance and genes encoding for the T6SS-1 that is related to inter-bacterial competition. In addition, we showed that this strain carries stx1c and stx2d act , a mucus-inducible variant. The results obtained in this study provide insights into STEC genomic plasticity and the importance of genomic islands in the adaptation and pathogenesis of this pathogen., (© 2022. Author(s).)

Background: Little is known about the evolution of peripheral arterial disease (PAD) since diagnosis and its association with glycemic and lipid control in patients with Type 2 Diabetes Mellitus (T2DM)., Objective: Evaluate the actual criteria to start screening PAD with ankle-brachial index (ABI) in T2DM patients and assess its progression and relationship with glycemic and lipid control since diagnosis., Methods: We performed a 3-year prospective cohort study with two groups: group 1 (978 individuals with T2DM undergoing drug treatment) and group 2 [221 newly diagnosed drug-naive (< 3 months) patients with T2DM]. PAD diagnosis was by ABI ≤ 0.90, regardless any symptoms., Results: As expected, abnormal ABI prevalence was higher in group 1 vs. Group 2 (87% vs. 60%, p < 0.001). However, abnormal ABI prevalence did not differ between patients over and under 50 years in both groups. Our drug-naive group stabilizes ABI (0.9 ± 0.1 vs 0.9 ± 0.1, p = NS) and improved glycemic and lipid control during follow-up [glycated hemoglobin (HbA1c) = 8.9 ± 2.1 vs 8.4 ± 2.3%, p < 0.05; LDL = 132 ± 45 vs 113 ± 38 mg/dL, p < 0.01, respectively]. When compared, patients who evolved with normalization or maintained normal ABI levels at the end [Group A, N = 60 (42%)] with those who decreased ABI to abnormal levels (ABI basal 1.0 ± 0.1 vs final 0.85 ± 0.1, p < 0.001) [Group B, N = 26 (18%)], an improvement in HbA1c (9 ± 2 vs 8 ± 2%, p < 0.05) and a correlation between the final HbA1c with ABI (r = - 0.3, p = 0.01) was found only in the first. In addition, a correlation was found between albuminuria variation and ABI solely in group A (r = - 0.3; p < 0.05)., Conclusion: Our study suggests that ABI should be measured at diagnosis in T2DM patients, indicating that current criteria to select patients to screen PAD with ABI must be simplified. An improvement in albuminuria and glycemic and lipid control could be related with ABI normalization in newly diagnosed T2DM drug-naive patients., (© 2022. The Author(s).)

Introduction. Candida spp. are commensal fungal pathogens of humans, but when there is an imbalance in the microbiota, or weak host immunity, these yeasts can become pathogenic, generating high medical costs. Gap Statement. With the increase in resistance to conventional antifungals, the development of new therapeutic strategies is necessary. This study evaluated the in vitro antifungal activity of chlorogenic acid against fluconazole-resistant strains of Candida spp. Mechanism of action through flow cytometry and in silico analyses, as well as molecular docking assays with ALS3 and SAP5, important proteins in the pathogenesis of Candida albicans associated with the adhesion process and biofilm formation. Results. The chlorogenic acid showed in vitro antifungal activity against the strains tested, causing reduced cell viability, increased potential for mitochondrial depolarization and production of reactive oxygen species, DNA fragmentation and phosphatidylserine externalization, indicating an apoptotic process. Concerning the analysis through docking, the complexes formed between chlorogenic acid and the targets Thymidylate Kinase , CYP51, 1 Yeast Cytochrome BC1 Complex e Exo -B-(1,3)- glucanase demonstrated more favourable binding energy. In addition, chlorogenic acid presented significant interactions with the ALS3 active site residues of C. albicans, important in the adhesion process and resistance to fluconazole. Regarding molecular docking with SAP5, no significant interactions were found between chlorogenic acid and the active site of the enzyme. Conclusion. We concluded that chlorogenic acid has potential use as an adjuvant in antifungal therapies, due to its anti- Candida activity and ability to interact with important drug targets.

Background: Type 1 Diabetes Mellitus (T1DM) impacts health-related quality of life (HRQoL). Cross-sectional studies suggest that low levels of vitamin D (VD) may impair HRQoL, however, the effect of VD supplementation on quality of life in T1DM patients has not yet been clarified. Our study evaluated the effects of high-dose VD supplementation on HRQoL in T1DM., Methods: We performed a prospective study with 64 patients receiving cholecalciferol (4000 IU/day for patients with 25-OH-vitamin D [25(OH)D] between 30 and 60 ng/mL, and 10,000 IU/day for those with 25(OH)D below 30 ng/mL) for 12 weeks, as part of a research protocol. HRQoL was assessed with EuroQol instruments (EQ-5D and EQ-VAS)., Results: There was an improvement in global EQ-5D index, and analysing specifically the EQ-5D domains, we observed an improvement in mobility (1.3 ± 0.6 versus 1.1 ± 0.3, p < 0.01). Evaluating possible outcome influencing variables, we detected a reduction in albuminuria at the end of the trial, without changes in BMI, lipids, blood pressure, glycemic control and insulin doses. We found correlations between final albuminuria and the dimensions: mobility (r = 0.6; p < 0.01), personal care (r = 0.7; p < 0.01), pain and discomfort (r = 0.6; p < 0.01) and habitual activities (r = 0.6; p < 0.01), suggesting an association between albuminuria reduction and the impact of VD supplementation on HRQoL., Conclusion: Our data showed that high doses of cholecalciferol supplementation can improve HRQoL in patients with T1DM, and the reduction of albuminuria seems to be an important factor in this context., Trial Registration: (ISRCTN32601947), 03/06/2017 retrospectively registered., (© 2022. The Author(s).)

Background: The detection of spatiotemporal clusters of deaths by coronavirus disease 2019 (COVID-19) is essential for health systems and services, as it contributes to the allocation of resources and helps in effective decision making aimed at disease control and surveillance. Thus we aim to analyse the spatiotemporal distribution and describe sociodemographic and clinical and operational characteristics of COVID-19-related deaths in a Brazilian state., Methods: A descriptive and ecological study was carried out in the state of Maranhão. The study population consisted of deaths by COVID-19 in the period from 29 March to 31 July 2020. The detection of spatiotemporal clusters was performed by spatiotemporal scan analysis., Results: A total of 3001 deaths were analysed with an average age of 69 y, predominantly in males, of brown ethnicity, with arterial hypertension and diabetes, diagnosed mainly by reverse transcription polymerase chain reaction in public laboratories. The crude mortality rates the municipalities ranged from 0.00 to 102.24 deaths per 100 000 inhabitants and three spatiotemporal clusters of high relative risk were detected, with a mortality rate ranging from 20.25 to 91.49 deaths per 100 000 inhabitants per month. The headquarters was the metropolitan region of São Luís and municipalities with better socio-economic and health development., Conclusions: The heterogeneous spatiotemporal distribution and the sociodemographic and clinical and operational characteristics of deaths by COVID-19 point to the need for interventions., (© The Author(s) 2021. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene.)

Background: Some authors evaluated the effect of VD on hyperglycemia in T1DM, but the results remain controversial. This study aims to analyze the effects of high-dose VD supplementation on T1DM patients' glycemic levels, maintaining stable doses of insulin., Methods: Prospective, 12-week clinical trial including 67 T1DM patients, supplemented with high doses of cholecalciferol according to participants' VD value. Patients with VD levels below 30 ng/mL received 10,000 IU/day; those with levels between 30-60 ng/mL received 4,000 IU/day. Patients who had not achieved 25(OH)D levels > 30 ng/ml or presented insulin dose variation during the study were not analyzed., Results: Only 46 out of 67 patients accomplished the criteria at the end of the study. There was no general improvement in the glycemic control evaluated by HbA1c (9.4 ± 2.4 vs 9.4 ± 2.6, p=NS) after VD supplementation. However, a post-hoc analysis, based on HbA1c variation, identified patients who had HbA1c reduced at least 0.6% (group 1, N = 13 (28%)). In addition, a correlation between 25(OH)D levels with HbA1c and total insulin dose at the end of the study was observed (r = -0.3, p<0.05; r=-0.4, p<0.05, respectively), and a regression model demonstrated that 25(OH)D was independent of BMI, duration of T1DM and final total insulin dose, being capable of determining 9.2% of HbA1c final levels (Unstandardized B coefficient = -0.033 (CI 95%: -0.064 to -0.002), r2 = 0.1, p <0.05)., Conclusion: Our data suggest that VD is not widely recommended for glycemic control. Nevertheless, specific patients might benefit from this approach., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background: The effect of glycemic control on diabetic kidney disease (DKD) is well known. Recent evidence has suggested that Vitamin D (VD) may have a nephroprotective effect in diabetes, but the relationship between VD, glycemic control, and albuminuria has yet to be clarified., Objective: Evaluate the relationship between 25-hydroxy-vitamin D [25(OH)D], HbA1c, and albuminuria in Diabetes Mellitus (DM)., Patients and Methods: Cross-sectional study with 1576 individuals with DM who had 25(OH)D, HbA1c, and albuminuria levels measured. Patients with abnormal creatinine levels were excluded, in order to avoid interference on VD levels by impaired kidney function., Results: Patients with HbA1c ≥7% had lower 25(OH)D when compared to patients with HbA1c <7% (29.7 ± 10.2 vs 28.1 ± 9.9 ng/ml, p = 0.003) and 25(OH)D levels seems to predict 1.5% of HbA1c behavior. The 25(OH)D concentrations in patients with normoalbuminuria were higher than the levels observed in those with micro or macroalbuminuria (29.8 ± 9.0 vs 26.8 ± 8.6 and 25.1 ± 7.6, respectively, p = 0.001), patients who had 25(OH)D <20 ng/ml and 25(OH)D <30 ng/ml were at a higher risk of presenting albuminuria [OR = 2.8 (95% CI = 1.6 - 4.9), p<0.001, and OR = 2.1 (95% CI = 1.3 - 4.6), p<0.001, respectively]. In our regression model, albuminuria was influenced by HbA1c (r² = 0.076, p<0.00001) and 25(OH)D (r² = 0.018, p = 0.002) independently., Conclusion: Our study found an association between vitamin D levels, HbA1c and DKD. Additionally, our data suggest that the association between urinary albumin excretion and vitamin D levels is independent of glycemic control in patients with diabetes. Even though our patients presented normal creatinine levels, it is necessary further prospective studies to confirm if this association precedes or not the loss of renal function., 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 © 2021 Felício, de Rider Britto, Cortez, de Souza Resende, de Lemos, de Moraes, de Aquino, de Souza Parente, de Queiroz, Abrahão Neto, de Alcântara, da Silva, de Souza Neto, Freire Piani, de Souza, Silva, de Oliveira, Said, Nascimento de Lemos, de Melo, Gomes, Contente Braga de Souza, de Sá Oliveira dos Reis, Leal, Lobato and Felício.)

Introduction: While soy is suggested as a possible risk factor, exclusive breastfeeding (EBF) has a likely protective effect in precocious puberty. Our aim was to evaluate the association between both of these variables with central precocious puberty (CPP)., Methods: We performed a retrospective, case-control study. A total of 161 girls were divided into two groups: 84 patients diagnosed with CPP composed the case group and 77 patients without the diagnosis of CPP (had gone through normal onset of puberty) were the control group., Results: Our control group had a higher presence of EBF >6 months, which was an important protective factor for CPP (OR: 0.5; IC 95%: 0.3-0.9, p = 0.05) and also correlated negatively with the presence of it (r = -0.2; p < 0.05). Oppositely, the use of soy was significantly higher in the CPP group, (OR: 3.8; IC 95%: 1.5-6, p < 0.05) and positively correlating (r = 0.2; p < 0.01) with the presence of CPP. Duration of soy intake (years) correlated with bone age (r = 0.415; p < 0.05). A logistic regression was performed to evaluate the effects of EBF duration and soy on CPP. The model was significant (x² (2) = 20,715, p = <0.001) and explained 12.2% (Nagelkerke R2) of the variance, correctly classifying 62.5% of cases. EBF was associated with a reduction of likelihood of having CPP [OR = 0,187 (CI = 0.055-0,635); Wald = 7,222, p = 0.007], while soy intake increased the risk [OR = 3.505 (CI) = 1,688-7,279, Wald = 11,319, p = 0.001]., Conclusion: Our data found the use of soy was associated with CPP. Additionally, EBF was pointed as a protective factor. However, future prospective studies are needed to clarify this issue., 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 © 2021 Felício, de Alcântara, Janaú, de Moraes, de Oliveira, de Lemos, de Souza Neto, Neto, da Silva, de Souza, Said, de Lemos, Vieira, Khayat, Santos, de Queiroz, de Sousa, Santos, de Melo, Piani and Felício.)

Staphylococcus aureus is a commensal bacterium and opportunistic human pathogen that can cause a wide variety of clinical infections. It is recognized for its ability to acquire antimicrobial resistance, so methicillin-resistant Staphylococcus aureus (MRSA) infections are a global healthcare challenge. Therefore, the development of new therapeutic options and alternative therapies for treatment is necessary. Curcumin, a polyphenolic substance found in the rhizome of turmeric longa L, has been shown to have several therapeutic properties, including antimicrobial activity. The objective of the study was to evaluate the in vitro antibacterial activity of curcumin alone and associated with oxacillin against MRSA strains, to analyze the mechanism of cell death involved in the isolated action of curcumin by means of flow cytometry and molecular docking, and to verify its superbiofilm action. Curcumin showed antibacterial activity in the range of 125-500 μg/mL against the tested strains, since it caused an increase in membrane permeability and DNA fragmentation, as revealed by flow cytometry analysis. Moreover, it was possible to observe interactions of curcumin with wild-type S. aureus DHFR, S. aureus gyrase and S. aureus gyrase complex with DNA, DNA (5'-D(*CP*GP*AP*TP*GP*CP*G)-3') and Acyl-PBP2a from MRSA by molecular docking. Curcumin also had a synergistic and additive effect when associated with oxacillin, and significantly reduced the cell viability of the analyzed biofilms. Thus, curcumin is a possible candidate for pharmaceutical formulation development for the treatment of MRSA infections., (Copyright © 2021. Published by Elsevier Ltd.)

The Dendrocephalus brasiliensis, a native species from South America, is a freshwater crustacean well explored in conservational and productive activities. Its main characteristics are its rusticity and resistance cysts production, in which the hatching requires a period of dehydration. Independent of the species utilization nature, it is essential to manipulate its cysts, such as the counting using microscopes. Manually counting is a difficult task, prone to errors, and that also very time-consuming. In this paper, we propose an automatized approach for the detection and counting of Dendrocephalus brasiliensis cysts from images captured by a digital microscope. For this purpose, we built the DBrasiliensis dataset, a repository with 246 images containing 5141 cysts of Dendrocephalus brasiliensis. Then, we trained two state-of-the-art object detection methods, YOLOv3 (You Only Look Once) and Faster R-CNN (Region-based Convolutional Neural Networks), on DBrasiliensis dataset in order to compare them under both cyst detection and counting tasks. Experiments showed evidence that YOLOv3 is superior to Faster R-CNN, achieving an accuracy rate of 83,74%, R2 of 0.88, RMSE (Root Mean Square Error) of 3.49, and MAE (Mean Absolute Error) of 2.24 on cyst detection and counting. Moreover, we showed that is possible to infer the number of cysts of a substrate, with known weight, by performing the automated counting of some of its samples. In conclusion, the proposed approach using YOLOv3 is adequate to detect and count Dendrocephalus brasiliensis cysts. The DBrasiliensis dataset can be accessed at: https://doi.org/10.6084/m9.figshare.13073240., Competing Interests: The authors have declared that no competing interests exist.

The genus Candida spp. has been highlighted as one of the main etiological agents causing fungal infections, with Candida albicans being the most prominent, responsible for most cases of candidemia. Due to its capacity for invasion and tissue adhesion, it is associated with the formation of biofilms, mainly in the environment and hospital devices, decreasing the effectiveness of available treatments. The repositioning of drugs, which is characterized by the use of drugs already on the market for other purposes, together with molecular-docking methods can be used aiming at the faster development of new antifungals to combat micro-organisms. This study aimed to evaluate the antifungal effect of diazepam on mature C. albicans biofilms in vitro and its action on biofilm in formation, as well as its mechanism of action and interaction with structures related to the adhesion of C. albicans , ALS3 and SAP5. To determine the MIC, the broth microdilution test was used according to protocol M27-A3 (CLSI, 2008). In vitro biofilm formation tests were performed using 96-well plates, followed by molecular-docking protocols to analyse the binding agent interaction with ALS3 and SAP5 targets. The results indicate that diazepam has antimicrobial activity against planktonic cells of Candida spp. and C. albicans biofilms, interacting with important virulence factors related to biofilm formation (ALS3 and SAP5). In addition, treatment with diazepam triggered a series of events in C. albicans cells, such as loss of membrane integrity, mitochondrial depolarization and increased production of EROs, causing DNA damage and consequent cell apoptosis.

Caseous lymphadenitis (CLA) is a chronic disease that affects small ruminants and causes economic losses in the associated breeding system. The causative agent of CLA is Corynebacterium pseudotuberculosis , a Gram-positive bacterium that exhibits tropism for external and internal lymph nodes and induces abscess formation in the host. Bacterial communities often produce a biofilm matrix that serves various functions, including protection against hostile environmental conditions, antibiotics, and the host immune response. Although biofilm formation has been reported for C. pseudotuberculosis , not all strains demonstrate this property in culture. In this work, we report the first comparative proteomic analysis of one biofilm-forming (CAPJ4) and one biofilm-non-forming strain (CAP3W) of C. pseudotuberculosis isolated from goats. Bacterial whole cell protein extracts were obtained for mass spectrometry analyses. Using LC-MS/MS, our studies reveal three and four proteins exclusively found in the CAPJ4 and CAP3W proteome, respectively. In addition, label-free quantitative analysis identified 40 proteins showing at-least 2-fold higher values in CAPJ4 compared CAP3W proteome Notably, CAPJ4 differentially synthesized the penicillin-binding protein, which participates in the formation of peptidoglycans. CAPJ4 also exhibited upregulation of N-acetylmuramoyl-L-alanine amidase and galactose-1-phosphate uridylyltransferase, which are involved in biofilm formation and exopolysaccharide biosynthesis. Here, we demonstrate that biofilm formation in C. pseudotuberculosis is likely associated with specific proteins, some of which were previously shown to be associated with virulence and biofilm formation in other organisms. Our findings may drive studies related to the bacterial mechanisms involved in the biofilm formation, in addition to providing targets for the treatment of CLA., 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 © 2021 Sá, Silva, Rodrigues, Rezende, Marchioro, Rocha Filho, Sousa, de Oliveira, Costa, Figueiredo, Portela, Castro, Azevedo, Seyffert and Meyer.)

The zoonotic enterohemorrhagic Escherichia coli (EHEC) O157: H7 bacterium causes diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS) in humans. Cattle are primary reservoirs and EHEC O157: H7; the bacteria predominately inhabit the colon and recto-anal junctions (RAJ). The early innate immune reactions in the infected gut are critical in the pathogenesis of EHEC O157: H7. In this study, calves orally inoculated with EHEC O157: H7 showed infiltration of neutrophils in the lamina propria of ileum and RAJ at 7 and 14 days post-infection. Infected calves had altered mucin layer and mast cell populations across small and large intestines. There were differential transcription expressions of key bovine β defensins, tracheal antimicrobial peptide (TAP) in the ileum, and lingual antimicrobial peptide (LAP) in RAJ. The main Gram-negative bacterial/LPS signaling Toll-Like receptor 4 (TLR4) was downregulated in RAJ. Intestinal infection with EHEC O157: H7 impacted the gut bacterial communities and influenced the relative abundance of Negativibacillus and Erysipelotrichaceae in mucosa-associated bacteria in the rectum. Thus, innate immunity in the gut of calves showed unique characteristics during infection with EHEC O157: H7, which occurred in the absence of major clinical manifestations but denoted an active immunological niche.

Background: Cardiovascular autonomic neuropathy (CAN) is associated with diabetes mellitus , increasing morbidity and mortality. Some cross-sectional studies associated CAN with low 25-hydroxyvitamin D levels. The aim of our study was to evaluate the effect of high-dose vitamin D (VD) supplementation on CAN in Type 1 Diabetes Mellitus (T1DM) patients., Methods: We performed a prospective study with 23 patients diagnosed with T1DM and CAN. Subjects with VD levels <30 ng/ml received 10,000 IU/day; the ones with VD levels between 30-60 ng/ml were given 4,000 IU/day for 12 weeks., Results: There was an improvement in CAN parameters related to resting heart rate variability, such as time domain parameters [Maximum RR interval (0.77 ± 0.11 vs 0.94 ± 0.51 s, p <0.05), Mean length of regular RR intervals (0.71 ± 0.10 vs 0.76 ± 0.09 s, p <0.05) and Standard deviation of all NN intervals (0.02 ± 0.01 vs 0.03 ± 0.02 s; p <0.01)] and frequency domain parameters [Low Frequency (1.9 ± 0.5 vs 2.5 ± 0.9 s, p < 0.001), Total Power (2.5 ± 0.4 vs 2.8 ± 0.6 s, p <0.05)]. In addition, there was a correlation between absolute VD level variation and posttreatment High Frequency (%), as well as among percent variation in VD level and end-of-study Low Frequency/High Frequency ratio (r=0.6, p<0.01; r= -0.5, p<0.05, respectively)., Conclusion: Our pilot study is the first to suggest a strong association between high-dose vitamin D supplementation and improved cardiovascular autonomic neuropathy in T1DM patients. It occurred without any variation in HbA1C, blood pressure levels, lipids, and insulin dose., Clinical Trial Registration: http://www.isrctn.com/ISRCTN32601947, identifier ISRCTN32601947., (Copyright © 2020 Silva, de Queiroz, de Melo, Abrahão Neto, Janaú, de Souza Neto, de Lemos, de Oliveira, de Alcântara, de Moraes, da Silva, de Souza, Said, de Lemos, Felício, Santos, Motta, dos Reis, Lobato, de Figueiredo, de Souza, Piani and Felício.)

Boron nitride nanotubes (BNNTs) have been growing in notoriety in the development of systems aiming bioapplications. In this work we conducted an investigation about the mechanisms involved in the incorporation of samarium and gadolinium in BNNTs. The process was performed by the reduction of samarium and gadolinium oxides (Sm 2 O 3 and Gd 2 O 3 , respectively) in the presence of NH 3 gas (witch decomposes into N 2 and H 2 ) at high temperatures. Various characterization techniques were conducted to elucidate how Sm and Gd are introduced into the BNNT structure. Biological in vitro assays were performed with human fibroblasts and a human osteosarcoma cell line (SAOS-2). Our results show that the studied systems have high potential for biomedical application and can be used as non-invasive imaging agents, such as scintigraphy radiotracers or as magnetic resonance imaging (MRI) contrast medium, being able to promote the treatment of many types of tumors simultaneously to their diagnosis., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interests, financial, scientific or otherwise in the publication of this article., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Purpose: Serum IGF-1 (Insulin like growth factor 1) and Growth Hormone (GH) provocative tests are reasonable tools for screening and diagnosis of idiopathic GH Deficiency (IGHD). However, the average cut-off points applied on these tests have a lower level of evidence and produce large amounts of false results. The aim of this study is to evaluate the sensitivity, specificity, and accuracy of IGF-1 and GH stimulation tests as diagnostic tools for IGHD, using clinical response to recombinant human GH (rhGH) treatment as diagnostic standard [increase of at least 0.3 in height standard deviation (H-SD) in 1 year]. Methods: We performed a prospective study with 115 children and adolescents presenting short stature (SS), without secondary SS etiologies such as organic lesions, genetic syndromes, thyroid disorders. They were separated into Group 1 [patients with familial SS or constitutional delay of growth and puberty (CDGP), not treated with rhGH], Group 2 (patients with suspicion of IGHD with clinical response to rhGH treatment), and Group 3 (patients with suspicion of IGHD without growth response to rhGH treatment). Then, they were assessed for diagnostic performance of IGF-1, Insulin Tolerance Test (ITT) and clonidine test (CT) alone and combined at different cut-off points. Results: Based on the ROC curve, the best cut-off points found for IGF-1, ITT, and CT when they were used isolated were -0.492 SDS (sensitivity: 50%; specificity: 53.8%; accuracy: 46.5%), 4.515 μg/L (sensitivity: 75.5%; specificity: 45.5%; accuracy: 52.7%), and 4.095 μg/L (sensitivity: 54.5%; specificity: 52.6%; accuracy: 56.9%), respectively. When we had combined IGF-1 with-2SD as cut-off alongside ITT or CT, we found 7 μg/L as the best cut-off point. In this situation, ITT had sensitivity, specificity and accuracy of 93.9, 81.8, and 90.1%, while CT had 93.2, 68.4, and 85.7%, respectively. Conclusion: Our data suggest that diagnosis of IGHD should be established based on a combination of clinical expertise, auxologic, radiologic, and laboratorial data, using IGF-1 at the -2SD threshold combined, with ITT or CT at the cut-off point of 7 μg/L. Additional studies, similar to ours, are imperative to establish cut-off points based on therapeutic response to rhGH in IGHD, which would be directly related to a better treatment outcome., (Copyright © 2019 Felício, Janaú, Moraes, Zahalan, de Souza Resende, de Lemos, de Souza Neto, Farias de Franco, Leitão, Silva, de Oliveira, de Alcântara, Contente Braga de Souza, da Silva, dos Santos, de Queiroz, de Moraes, de Figueiredo, Farinassi, Farias, da Silva, Felício and Abrahão Neto.)

Human pathogenic gram-negative bacteria, such as enteropathogenic Escherichia coli (EPEC), rely on type III secretion systems (T3SS) to translocate virulence factors directly into host cells. The coiled-coil domains present in the structural proteins of T3SS are conformed by amphipathic alpha-helical structures that play an important role in the protein-protein interaction and are essential for the assembly of the translocation complex. To investigate the inhibitory capacity of these domains on the T3SS of EPEC, we synthesized peptides between 7 and 34 amino acids based on the coiled-coil domains of proteins that make up this secretion system. This analysis was performed through in vitro hemolysis assays by assessing the reduction of T3SS-dependent red blood cell lysis in the presence of the synthesized peptides. After confirming its inhibitory capacity, we performed molecular modeling assays using combined techniques, docking-molecular dynamic simulations, and quantum-mechanic calculations of the various peptide-protein complexes, to improve the affinity of the peptides to the target proteins selected from T3SS. These techniques allowed us to demonstrate that the peptides with greater inhibitory activity, directed against the coiled-coil domain of the C-terminal region of EspA, present favorable hydrophobic and hydrogen bond molecular interactions. Particularly, the hydrogen bond component is responsible for the stabilization of the peptide-protein complex. This study demonstrates that compounds targeting T3SS from pathogenic bacteria can indeed inhibit bacterial infection by presenting a higher specificity than broad-spectrum antibiotics. In turn, these peptides could be taken as initial structures to design and synthesize new compounds that mimic their inhibitory pharmacophoric pattern., (© 2019 European Peptide Society and John Wiley & Sons, Ltd.)

Background: Streptococcus agalactiae (GBS) is a major pathogen of Nile tilapia, a global commodity of the aquaculture sector. The aims of this study were to evaluate protein expression in the main genotypes of GBS isolated from diseased fishes in Brazil using a label-free shotgun nano-liquid chromatography-ultra definition mass spectrometry (nanoLC-UDMS E ) approach and to compare the differential abundance of proteins identified in strains isolated from GBS-infected fishes and humans., Results: A total of 1070 protein clusters were identified by nanoLC-UDMS E in 5 fish-adapted GBS strains belonging to sequence types ST-260 and ST-927 and the non-typeable (NT) lineage and 1 human GBS strain (ST-23). A total of 1065 protein clusters corresponded to the pan-proteome of fish-adapted GBS strains; 989 of these were identified in all fish-adapted GBS strains (core proteome), and 62 were shared by at least two strains (accessory proteome). Proteins involved in the stress response and in the regulation of gene expression, metabolism and virulence were detected, reflecting the adaptive ability of fish-adapted GBS strains in response to stressor factors that affect bacterial survival in the aquatic environment and bacterial survival and multiplication inside the host cell. Measurement of protein abundance among different hosts showed that 5 and 26 proteins were exclusively found in the human- and fish-adapted GBS strains, respectively; the proteins exclusively identified in fish isolates were mainly related to virulence factors. Furthermore, 215 and 269 proteins were up- and down-regulated, respectively, in the fish-adapted GBS strains in comparison to the human isolate., Conclusions: Our study showed that the core proteome of fish-adapted GBS strains is conserved and demonstrated high similarity of the proteins expressed by fish-adapted strains to the proteome of the human GBS strain. This high degree of proteome conservation of different STs suggests that, a monovalent vaccine may be effective against these variants.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a human pathogen responsible for diarrhea, hemorrhagic colitis and hemolytic uremic syndrome (HUS). To promote a comprehensive insight into the molecular basis of EHEC O157:H7 physiology and pathogenesis, the combined proteome of EHEC O157:H7 strains, Clade 8 and Clade 6 isolated from cattle in Argentina, and the standard EDL933 (clade 3) strain has been analyzed. From shotgun proteomic analysis a total of 2,644 non-redundant proteins of EHEC O157:H7 were identified, which correspond approximately 47% of the predicted proteome of this pathogen. Normalized spectrum abundance factor analysis was performed to estimate the protein abundance. According this analysis, 50 proteins were detected as the most abundant of EHEC O157:H7 proteome. COG analysis showed that the majority of the most abundant proteins are associated with translation processes. A KEGG enrichment analysis revealed that Glycolysis / Gluconeogenesis was the most significant pathway. On the other hand, the less abundant detected proteins are those related to DNA processes, cell respiration and prophage. Among the proteins that composed the Type III Secretion System, the most abundant protein was EspA. Altogether, the results show a subset of important proteins that contribute to physiology and pathogenicity of EHEC O157:H7., Competing Interests: The authors have declared that no competing interests exist.

Sensorineural hearing impairment has been associated with DM, and it is probably linked to the same pathophysiological mechanisms as well-established in microvascular diabetes complications. The study of otoacoustic emissions (OAEs) is useful to identify subclinical cochlear dysfunction. Therefore, the aim of this study was to evaluate the association between abnormal OAEs responses, diabetic kidney disease (DKD) and diabetic cardiac autonomic neuropathy (CAN). We performed a cross-sectional study with 37 type 1 DM patients without auditory symptoms, submitted to the study of Distortion Product Otoacoustic Emissions (DPOAEs) and screened for DKD and CAN. The otoacoustic emissions responses were considered abnormal in 27/37 (73%) patients. A correlation was found between abnormal OAEs responses and presence of DKD (r = 0.36, p < 0.05), and 14/16 (88%) patients with a lower amplitude of OAEs in 8 kHz frequency band presented DKD. Abnormal OAEs responses in the 6 kHz frequency band were correlated with the presence (r = 0.41, p = 0.01) and severity of CAN (r = 0.44, p < 0.001). Additionally, 7/9 (78%) patients with abnormal OAE responses in this frequency also presented abnormal CAN scores. Our results suggest that abnormal otoacoustic emissions responses in high frequency bands are associated with diabetes microvascular complications and could be a risk marker for DKD and CAN, presenting low sensitivity and high specificity. Therefore, assuming that hearing impairment is a pre-clinical stage of hearing loss, performing distortion product otoacoustic emissions in T1DM patients with microvascular complications could be useful to identify those who would be benefit with regular audiologic follow up and tighter diabetes control.

The development of a myriad of nanoparticles types has opened new possibilities for the diagnostics and treatment of many diseases, especially for cancer. However, most of the researches done so far do not focus on the protection of normal cells surrounding a tumor from irradiation bystander effects that might lead to cancer recurrence. Gap-junctions are known to be involved in this process, which leads to genomic instability of neighboring normal cells, and flufenamic acid (FFA) is included in a new group of gap-junction blockers recently discovered. The present work explores the use of mesoporous silica nanoparticles MCM-41 functionalized with 3-Aminopropyltriethoxysilane (APTES) for anchoring the flufenamic acid for its prolonged and controlled release and protection from radiation bystander effects. MCM-41 and functionalized samples were structurally and chemically characterized with multiple techniques. The biocompatibility of all samples was tested in a live/dead assay performed in cultured MRC-5 and HeLa cells. HeLa cells cultured were exposed to 50 Gy of gamma-rays and the media transferred to fibroblast cells cultured separately. Our results show that MCM-41 and functionalized samples have high biocompatibility with MCR-5 and HeLa cells, and most importantly, the FFA delivered by these NPs was able to halt apoptosis, one of main bystander effects.

Boron nitride nanotubes doped in situ with samarium (Sm-doped BNNTs) were synthesized at 1150°C under atmosphere of NH 3 /N 2 gas mixture by thermal chemical vapor deposition (TCVD) using samarium oxide that is a product of the process separation of thorium and uranium tailings. The samarium in the BNNTs sample was activated by neutron capture, in a nuclear reactor, producing 152 Sm radioisotopes. The STEM-EELS spectrum and neutron activation show energies attributed to the samarium confirming the in situ doping process during BNNTs growth. The results demonstrate that this material has great potential as a nanosized β - emission source for medical therapy., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Context: Cold-water immersion (CWI) has been applied widely as a recovery method, but little evidence is available to support its effectiveness., Objective: To investigate the effects of CWI on muscle damage, perceived muscle soreness, and muscle power recovery of the upper and lower limbs after jiu-jitsu training., Design: Crossover study., Setting: Laboratory and field., Patients or Other Participants: A total of 8 highly trained male athletes (age = 24.0 ± 3.6 years, mass = 78.4 ± 2.4 kg, percentage of body fat = 13.1% ± 3.6%) completed all study phases., Intervention(s): We randomly selected half of the sample for recovery using CWI (6.0°C ± 0.5°C) for 19 minutes; the other participants were allocated to the control condition (passive recovery). Treatments were reversed in the second session (after 1 week)., Main Outcome Measure(s): We measured serum levels of creatine phosphokinase, lactate dehydrogenase (LDH), aspartate aminotransferase, and alanine aminotransferase enzymes; perceived muscle soreness; and recovery through visual analogue scales and muscle power of the upper and lower limbs at pretraining, postrecovery, 24 hours, and 48 hours., Results: Athletes who underwent CWI showed better posttraining recovery measures because circulating LDH levels were lower at 24 hours postrecovery in the CWI condition (441.9 ± 81.4 IU/L) than in the control condition (493.6 ± 97.4 IU/L; P = .03). Estimated muscle power was higher in the CWI than in the control condition for both upper limbs (757.9 ± 125.1 W versus 695.9 ± 56.1 W) and lower limbs (53.7 ± 3.7 cm versus 35.5 ± 8.2 cm; both P values = .001). In addition, we observed less perceived muscle soreness (1.5 ± 1.1 arbitrary units [au] versus 3.1 ± 1.0 au; P = .004) and higher perceived recovery (8.8 ± 1.9 au versus 6.9 ± 1.7 au; P = .005) in the CWI than in the control condition at 24 hours postrecovery., Conclusions: Use of CWI can be beneficial to jiu-jitsu athletes because it reduces circulating LDH levels, results in less perceived muscle soreness, and helps muscle power recovery at 24 hours postrecovery.

Ethanol production from acidic-alkaline pretreated cashew apple bagasse (CAB-OH) was investigated using separated hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) processes. First, a screening of Kluyveromyces strains was conducted by SHF and a maximum ethanol concentration of 24.1 g L(-1) was obtained using Kluyveromyces marxianus ATCC36907, which presented similar profiles when compared to results obtained by a Saccharomyces strain. The effect of temperature on ethanol production conducted by SHF using K. marxianus ATCC36907 was investigated, and the maximum ethanol yield (YE/G) was obtained at 40 °C (0.46 g g(-1)) using a synthetic medium. In the SHF using CAB-OH hydrolysate, the maximum ethanol concentration obtained was 24.9 g L(-1), 5.92 g L(-1) h(-1) of productivity, and ethanol yield of 0.43 g g(-1) at 40 °C. Afterwards, K. marxianus ATCC36907 was used in the bioconversion of CAB-OH by SSF, and an ethanol concentration of 41.41 ± 0.2 g L(-1) was obtained using 10 % CAB-OH at 40 °C, 150 rpm and 24 h, resulting in a Y'E/G of 0.50 gE gG (-1) and an efficiency of 98.4 %, in the process conducted with cellobiase supplementation. SHF and SSF processes using CAB-OH and K. marxianus ATCC36907 can be used to ethanol production, but the SSF process required only one step to achieve the same production.

Introduction: The aim of this study was to identify the determinants of distance walked in six-minute walk test (6MWD) in patients undergoing cardiac surgery at hospital discharge., Methods: The assessment was performed preoperatively and at discharge. Data from patient records were collected and measurement of the Functional Independence Measure (FIM) and the Nottingham Health Profile (NHP) were performed. The six-minute walk test (6MWT) was performed at discharge. Patients undergoing elective cardiac surgery, coronary artery bypass grafting or valve replacement were eligible. Patients older than 75 years who presented arrhythmia during the protocol, with psychiatric disorders, muscular or neurological disorders were excluded from the study., Results: Sixty patients (44.26% male, mean age 51.53 ± 13 years) were assessed. In multivariate analysis the following variables were selected: type of surgery (P = 0.001), duration of cardiopulmonary bypass (CPB) (P = 0.001), Functional Independence Measure - FIM (0.004) and body mass index - BMI (0.007) with r = 0.91 and r2 = 0.83 with P < 0.001. The equation derived from multivariate analysis: 6MWD = Surgery (89.42) + CPB (1.60) + MIF (2.79 ) - BMI (7.53) - 127.90., Conclusion: In this study, the determinants of 6MWD in patients undergoing cardiac surgery were: the type of surgery, CPB time, functional capacity and body mass index.

Chromobacterium violaceum is a beta-proteobacterium with high biotechnological potential, found in tropical environments. This bacterium causes opportunistic infections in both humans and animals, that can spread throughout several tissues, quickly leading to the death of the host. Genomic studies identified potential mechanisms of pathogenicity but no further studies were done to confirm the expression of these systems. In this study 36 unique protein entries were identified in databank from a two-dimensional profile of C. violaceum secreted proteins. Chromobacterium violaceum exoproteomic preliminary studies confirmed the production of proteins identified as virulence factors (such as a collagenase, flagellum proteins, metallopeptidases, and toxins), allowing us to better understand its pathogenicity mechanisms. Biotechnologically interesting proteins (such as chitinase and chitosanase) were also identified among the secreted proteins, as well as proteins involved in the transport and capture of amino acids, carbohydrates, and oxidative stress protection. Overall, the secreted proteins identified provide us important insights on pathogenicity mechanisms, biotechnological potential, and environment adaptation of C. violaceum.

Lipidic α-amino acids (LAAs) have been described as non-natural amino acids with long saturated or unsaturated aliphatic chains. In the continuing prospect to discover anticancer agents from marine sources, we have obtained a mixture of two cytotoxic LAAs (1a and 1b) from the zoanthid Protopalythoa variabilis. The anti-proliferative potential of 14 synthetic LAAs and 1a/1b were evaluated on four tumor cell lines (HCT-8, SF-295, MDA-MB-435, and HL-60). Five of the synthetic LAAs showed high percentage of tumor cell inhibition, while 1a/1b completely inhibited tumor cell growth. Additionally, apoptotic effects of 1a/1b were studied on HL-60 cell line. 1a/1b-treated cells showed apoptosis morphology, loss of mitochondrial potential, and DNA fragmentation., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Two new labdane diterpenes, 6alpha-malonyloxy-ethyl ester manoyl oxide and bis-6alpha-dioxymanoylmalonate, together with the known 6alpha-hydroxymanoyl oxide, 6alpha-malonyloxymaloyl oxide and betulinic acid were isolated from leaves of Stemodia trifoliata. Their structures were elucidated by spectroscopic studies (IR and HR-ESI-MS), including an extensive NMR (COSY, HSQC, HMBC and NOESY) analysis., (Copyright (c) 2010 John Wiley & Sons, Ltd.)

In the present study, a novel, fast, sensitive and robust method to quantify ethinylestradiol in human plasma using 17alpha-ethinylestradiol-d4 as the internal standard (IS) is described. The analyte and the IS were extracted from acidified plasma by liquid-liquid extraction (LLE) using diethyl ether-hexane followed by online solid phase extraction (SPE) using online C18 cartridges. Extracted samples were analyzed by high-performance liquid chromatography coupled to atmospheric pressure photoionization tandem mass spectrometry (HPLC-APPI-MS/MS). Chromatography was performed isocratically on a C18, 5 microm analytical column. The method had a chromatographic run time of 2.50 min and a linear calibration curve over the range 5-500 pg ml(-1) (r(2)>0.9992). The lowest concentration quantified was 5 pg ml(-1), demonstrating acceptable accuracy and precision. The intra-assay precisions ranged from 2.1 to 14.6%, while inter-assay precisions ranged from 4.4 to 11.4%. The intra-assay accuracies ranged from 94.6 to 103.8%, while the inter-assay accuracies ranged from 98.9 to 101.6%. The recovery of ethinylestradiol was determined as part of the assay validation process and was 73.1 and 79.0% for the concentrations 15 and 375 pg ml(-1), respectively. Short-term stability showed that ethinylestradiol was stable in plasma for at least 19 h at room temperature or for at least 385 days when stored at -20 degrees C. In the study of bioequivalence conducted in Brazil, healthy volunteers received two ethinylestradiol 0.035 mg tablet formulations using an open, randomized, two-period crossover design with a 2-week washout interval. Since the 90% confidence interval for C(max) and area under the curve ratios were all inside the 80-125% interval proposed by the US Food and Drug Administration, it was concluded that the two ethinylestradiol formulations are bioequivalent with respect to both the rate and the extent of absorption.

The ciliate Trichodina was recorded on the calanoid Notodiaptomus deitersi in a shallow, eutrophic reservoir, located in the centre-west of Brazil. The species was confirmed as Trichodina diaptomi, a species widely distributed in Europe, Africa, Asia and Australia. It was observed moving freely over the carapace of the copepod, and using its adhesive disc. This is the first record for the species in the Americas.

A specific, fast and sensitive high performance liquid chromatography (HPLC) coupled to atmospheric pressure photoionization (APPI) tandem mass spectrometric (LC-MS/MS) assay was developed for the determination of cyproterone (CYP) acetate (CAS 427-51-0) in human plasma. The retention times were 3.26 and 2.90 min for CYP acetate and its internal standard (I. S.) finasteride (FIN), respectively. The overall mean recovery, using liquid/liquid extraction, was found to be 109.0, 107.7 and 100.3%, for low, medium and high concentrations, respectively. Calibration curves were linear in the concentration range of 0.1-50.0 ng/ml, and the lower limit of quantification (LLOQ) was 0.1 ng/ml. The LLOQ, 0.1 ng/ml, was sensitive enough for detecting terminal phase concentrations of the drug. Inter-batch precision of the method ranged from 2.2 to 5.55%, while Inter-batch accuracy ranged from 95.5 to 100.0%. Intra-batch precision ranged from 1.8 to 5.6%, while Intra-batch accuracy ranged from 92.0 to 99.4% at concentrations of 0.3 ng/ml, 20.0 and 40.0 ng/ml. The developed method was applied to a bioequivalenc study of CYP acetate in a group of 44 female volunteers at a single oral dose of a 2 mg tablet, in a combination of ethinylestradiol/CYP acetate (0.25/2 mg). The plasma concentration of CYP acetate did not differ significantly after administration of both formulations (test formulation and the reference one). The geometric mean and respective 90% CI of CYP acetate test/reference percent ratios were 90.66% (84.39-97.40%) for Cmax and 96.20% (90.45-102.33%) for AUC0-t.

Unlabelled: We investigated the effect of transcutaneous electrical nerve stimulation (TENS) for inguinal herniorrhaphy postoperative pain control in a prospective, randomized, double-blinded, placebo-controlled study. Forty patients undergoing unilateral inguinal herniorrhaphy with an epidural anesthetic technique were randomly allocated to receive either active TENS or placebo TENS. Postoperative pain was evaluated using a standard 10-point numeric rating scale (NRS). Analgesic requirements were also recorded. TENS (100 Hz, strong but comfortable sensory intensity) was applied for 30 minutes through 4 electrodes placed around the incision twice, 2 and 4 hours after surgery. Pain was assessed before and after each application of TENS and 8 and 24 hours after surgery. In the group treated with active TENS, pain intensity was significantly lower 2 hours (P = .028), 4 hours (P = .022), 8 hours (P = .006), and 24 hours (P = .001) after the surgery when compared with the group that received placebo TENS. Active TENS also decreased analgesic requirements in the postoperative period when compared with placebo TENS (P = .001). TENS is thus beneficial for postoperative pain relief after inguinal herniorrhaphy; it has no observable side effects, and the pain-reducing effect continued for at least 24 hours. Consequently, the routine use of TENS after inguinal herniorrhaphy is recommended., Perspective: This study presents the hypoalgesic effect of high-frequency TENS for postoperative pain after inguinal herniorrhaphy. This may reinforce findings from basic science showing an opioid-like effect provided by TENS, given that high-frequency TENS has been shown to activate delta-opioid receptors.