Evolutionary Dynamics of Accelerated Antiviral Resistance Development in Hypermutator Herpesvirus.

Antiviral therapy is constantly challenged by the emergence of resistant pathogens. At the same time, experimental approaches to understand and predict resistance are limited by long periods required for evolutionary processes. Here, we present a herpes simplex virus 1 mutant with impaired proofreading capacity and consequently elevated mutation rates. Comparing this hypermutator to parental wild type virus, we study the evolution of antiviral drug resistance in vitro. We model resistance development and elucidate underlying genetic changes against three antiviral substances. Our analyzes reveal no principle difference in the evolutionary behavior of both viruses, adaptive processes are overall similar, however significantly accelerated for the hypermutator. We conclude that hypermutator viruses are useful for modeling adaptation to antiviral therapy. They offer the benefit of expedited adaptation without introducing apparent bias and can therefore serve as an accelerator to predict natural evolution.

Suicidal Phenotype of Proofreading-Deficient Herpes Simplex Virus 1 Polymerase Mutants.

Herpes simplex virus 1 (HSV-1) encodes a family B DNA polymerase (Pol) capable of exonucleolytic proofreading whose functions have been extensively studied in the past. Early studies on the in vitro activity of purified Pol protein found that the enzymatic functions of the holoenzyme are largely separate. Consequently, exonuclease activity can be reduced or abolished by certain point mutations within catalytically important regions, with no or only minor effects on polymerase activity. Despite unimpaired polymerase activity, the recovery of HSV-1 mutants with a catalytically inactive exonuclease has been so far unsuccessful. Hence, mutations such as D368A, which abolish exonuclease activity, are believed to be lethal. Here, we show that HSV-1 can be recovered in the absence of Pol intrinsic exonuclease activity and demonstrate that a lack of proofreading causes the rapid accumulation of likely detrimental mutations. Although mutations that abolish exonuclease activity do not appear to be lethal, the lack of proofreading yields viruses with a suicidal phenotype that cease to replicate within few passages following reconstitution. Hence, we conclude that high replication fidelity conferred by proofreading is essential to maintain HSV-1 genome integrity and that a lack of exonuclease activity produces an initially viable but rapidly suicidal phenotype. However, stably replicating viruses with reduced exonuclease activity and therefore elevated mutation rates can be generated by mutating a catalytically less important site located within a conserved exonuclease domain. IMPORTANCE Recovery of fully exonuclease-deficient herpes simplex virus 1 (HSV-1) DNA polymerase mutants has been so far unsuccessful. However, exonuclease activity is not known to be directly essential for virus replication, and the lethal phenotype of certain HSV-1 polymerase mutants is thus attributed to factors other than exonuclease activity. Here, we showed that the recovery of a variety of exonuclease-deficient HSV-1 polymerase mutants is possible and that these mutants are initially replication competent. We, however, observed a progressive loss of mutant viability upon cell culture passaging, which coincided with the rapid accumulation of mutations in exonuclease-deficient viruses. We thus concluded that a lack of DNA proofreading in exonuclease-deficient viruses causes an initially viable but rapidly suicidal hypermutator phenotype and, consequently, the extinction of mutant viruses within few generations following recovery. This would make the absence of exonuclease activity the primary reason for the long-reported difficulties in culturing exonuclease-deficient HSV-1 mutants.

A non-transmissible live attenuated SARS-CoV-2 vaccine.

Live attenuated vaccines (LAVs) administered via the mucosal route may offer better control of the COVID-19 pandemic than non-replicating vaccines injected intramuscularly. Conceptionally, LAVs have several advantages, including presentation of the entire antigenic repertoire of the virus, and the induction of strong mucosal immunity. Thus, immunity induced by LAV could offer superior protection against future surges of COVID-19 cases caused by emerging SARS-CoV-2 variants. However, LAVs carry the risk of unintentional transmission. To address this issue, we investigated whether transmission of a SARS-CoV-2 LAV candidate can be blocked by removing the furin cleavage site (FCS) from the spike protein. The level of protection and immunity induced by the attenuated virus with the intact FCS was virtually identical to the one induced by the attenuated virus lacking the FCS. Most importantly, removal of the FCS completely abolished horizontal transmission of vaccine virus between cohoused hamsters. Furthermore, the vaccine was safe in immunosuppressed animals and showed no tendency to recombine in vitro or in vivo with a SARS-CoV-2 field strain. These results indicate that removal of the FCS from SARS-CoV-2 LAV is a promising strategy to increase vaccine safety and prevent vaccine transmission without compromising vaccine efficacy.

Engineering and Characterization of Avian Coronavirus Mutants Expressing Fluorescent Reporter Proteins from the Replicase Gene.

Infectious bronchitis virus (IBV) is an avian coronavirus that causes infectious bronchitis, an acute and highly contagious respiratory disease of chickens. IBV evolution under the pressure of comprehensive and widespread vaccination requires surveillance for vaccine resistance, as well as periodic vaccine updates. Reverse genetics systems are very valuable tools in virology, as they facilitate rapid genetic manipulation of viral genomes, thereby advancing basic and applied research. We report here the construction of an infectious clone of IBV strain Beaudette as a bacterial artificial chromosome (BAC). The engineered full-length IBV clone allowed the rescue of an infectious virus that was phenotypically indistinguishable from the parental virus. We used the infectious IBV clone and examined whether an enhanced green fluorescent protein (EGFP) can be produced by the replicase gene ORF1 and autocatalytically released from the replicase polyprotein through cleavage by the main coronavirus protease. We show that IBV tolerates insertion of the EGFP ORF at the 3' end of the replicase gene, between the sequences encoding nsp13 and nsp16 (helicase, RNA exonuclease, RNA endonuclease, and RNA methyltransferase). We further show that EGFP is efficiently cleaved from the replicase polyprotein and can be localized in double-membrane vesicles along with viral RNA polymerase and double-stranded RNA, an intermediate of IBV genome replication. One of the engineered reporter EGFP viruses were genetically stable during passage in cultured cells. We demonstrate that the reporter EGFP viruses can be used to study virus replication in host cells and for antiviral drug discovery and development of diagnostic assays. IMPORTANCE Reverse genetics systems based on bacterial artificial chromosomes (BACs) are the most valuable systems in coronavirus research. Here, we describe the establishment of a reverse genetics system for the avian coronavirus strain Beaudette, the most intensively studied strain. We cloned a copy of the avian coronavirus genome into a BAC vector and recovered infectious virus in permissive cells. We used the new system to construct reporter viruses that produce enhanced green fluorescent protein (EGFP). The EGFP coding sequence was inserted into 11 known cleavage sites of the major coronavirus protease in the replicase gene ORF1. Avian coronavirus tolerated the insertion of the EGFP coding sequence at three sites. The engineered reporter viruses replicated with parental efficiency in cultured cells and were sufficiently genetically stable. The new system facilitates functional genomics of the avian coronavirus genome but can also be used for the development of novel vaccines and anticoronaviral drugs.

Potential Therapeutic Targets of Quercetin in the Cutaneous Melanoma Model and Its Cellular Regulation Pathways: A Systematic Review.

Melanoma is a skin cancer with a high mortality rate due to its invasive characteristics. Currently, immunotherapy and targeted therapy increase patient survival but are ineffective in the advanced stages of the tumor. Quercetin (Que) is a natural compound that has demonstrated chemopreventive effects against different types of tumors. This review provides evidence for the therapeutic potential of Que in melanoma and identifies its main targets. The Scopus, Web of Science, and PubMed databases were searched, and studies that used free or encapsulated Que in melanoma models were included, excluding associations, analogs, and extracts. As a result, 73 articles were retrieved and their data extracted. Que has multiple cellular targets in melanoma models, and the main regulated pathways are cell death, redox metabolism, metastasis, and melanization. Que was also able to regulate important targets of signaling pathways, such as PKC, RIG-I, STAT, and P53. In murine models, treatment with Que reduced tumor growth and weight, and decreased metastatic nodules and angiogenic vasculature. Several studies have incorporated Que into carriers, demonstrating improved efficacy and delivery to tumors. Thus, Que is a promising therapeutic agent for the treatment of melanoma; however, further studies are needed to evaluate its effectiveness in clinical trials.

Diet supplemented with Saccharomyces cerevisiae from different fermentation media modulates the faecal microbiota and the intestinal fermentative products in dogs.

This study aimed at evaluating the coefficients of total tract apparent digestibility (CTTAD) of nutrients, metabolisable energy (ME), diet palatability, faecal fermentative products and microbiota of dogs fed yeasts from different fermentation media and its fractions. Four diets were evaluated: control, without yeast (CO); diet with 10 g/kg brewer's yeast (BY); diet with 10 g/kg brewer's yeast + corn yeast (BCY); and diet with 10 g/kg BCY + cell wall fractions (BCYF). Twelve adult dogs were distributed in a randomized block design (periods). Each of the four diets was fed to a group of three dogs per period of 20 days, totalling two periods and six repetitions per treatment. Sixteen adult dogs were used for the palatability test, which compared the CO diet versus each one of the yeast diets. Data with normal distribution were subjected to analysis of variance (p < 0.05). Means were compared by orthogonal contrasts (p < 0.05): (A) CO diet versus BY, BCY and BCYF diets; (B) BY diet versus BCY and BCYF diets; (C) BCY diet versus BCYF diet. There was no difference in the CTTAD and ME of the diets (p > 0.05). Yeast diets reduced faecal odour and indole peak area (p < 0.05). Faecal short-chain fatty acids concentration was greater in dogs fed yeast diets compared to those fed the CO (p < 0.05). Yeast diets showed a higher intake ratio compared to the CO (p < 0.05). The BCY and BCYF diets resulted in a greater abundance of Bacteroides, Faecalibacterium, Coprococcus, and Phascolarctobacterium in relation to the CO (p < 0.05). Our results suggest that dietary yeast supplementation results in beneficial changes in intestinal functionality indicators, mainly with the combination of yeasts from brewers and corn fermentation media. In addition, yeast supplementation improves diet palatability without compromising nutrient digestibility.

Clinical pharmaceutical screening in critical situations in a radioiodine therapy management service.

Radioiodine therapy can be used in differentiated thyroid carcinoma and requires extensive evaluation to ensure effectiveness and safety. Therefore, it is necessary to evaluate all health problems and medications used in the pre-radioiodine therapy period and comprehensive medication managementservices can serve as a screening tool in this context. The present study aims to describe critical clinical situations identified during the initial assessments of a comprehensive medication management service offered to differentiated thyroid carcinoma patients pre-radioiodine therapy, and the pharmaceutical interventions performed to solve them. A descriptive study with regard to the initial ten months of a comprehensive medication management service was carried out in a large oncology hospital (Rio de Janeiro, Brazil). Descriptive analysis was used to describe the critical clinical situations identified, as well as the correspondent drug therapy problems and the type, acceptability, and outcomes of the pharmaceutical interventions performed to solve them. Thirty patients with an average of 45.8 years and 5.1 medications were evaluated. Five critical clinical situations were identified; corresponding to drug therapy problems two(needs additional drug therapy - n = 4) and drug therapy problems four (dosage too low - n = 1). All pharmaceutical interventions were accepted. The comprehensive medication management service provision pre-radioiodine therapy is feasible and represents an important screening strategy.

Validation of a HPLC method for quantification of midazolam in rat plasma: Application during a Maytenus ilicifolia-drug interaction study.

Midazolam (MDZ) is routinely employed as a marker compound of cytochrome P450 3A (CYP3A) activity. Despite the many HPLC-UV methods described to quantify MDZ in plasma, all of them use acetonitrile (ACN) or a mixture of methanol-isopropanol as organic solvent of the mobile phase. Since the ACN shortage in 2008, efforts have been made to replace this solvent during HPLC analysis. A simple, sensitive, accurate and repeatable HPLC-UV method (220 nm) was developed and validated to quantify MDZ in rat plasma using methanol instead. The method was applied during a herb-drug interaction study involving Maytenus ilicifolia, a Brazilian folk medicine used to treat gastric disorders. Plasma samples were alkalinized and MDZ plus alprazolam (internal standard) were extracted with diethyl ether. After solvent removal, the residue was reconstituted with methanol-water (1:1). The analyte was eluted throughout a C18 column using sodium acetate buffer (10 mm, pH 7.4)-methanol (40:60, v/v). The precision at the lower limit of quantification never exceeded 19.40%, and 13.86% at the higher levels of quality control standards, whereas the accuracy ranged from -19.81 to 14.33%. The analytical curve was linear from 50 to 2,000 ng/ml. The activity of the hepatic CYP3A enzymes was not affected by the extract.

Evaluation of antibacterial and enhancement of antibiotic action by the flavonoid kaempferol 7-O-ß-D-(6″-O-cumaroyl)-glucopyranoside isolated from Croton piauhiensis müll.

There has been a rapid increase in the incidence and prevalence of opportunistic bacterial infections. Inappropriate use of current antibiotics has continuously contributed to the emergence of resistance to conventional antibiotic therapy. Therefore, the search for natural molecules that are able to combat infections is of great public interest, and many of these compounds with antimicrobial properties can be obtained from phytochemical studies of medicinal plants. In this context, this study reports the isolation and characterization of the flavonoid, kaempferol 7-O-ß-D-(6″-O-cumaroyl)-glucopyranoside, from Croton piauhiensis leaves. Additionally, the intrinsic antimicrobial action of the compound and its enhancement against Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus strains were assessed. The minimum inhibitory concentration (MIC) of the compound was determined using broth microdilution assays. To evaluate the modulatory effect of the flavonoid, the MIC of antibiotics amikacin and gentamicin, belonging to the class aminoglycosides was assessed, with and without the compound in sterile microplates. The results of intrinsic antibacterial activity tests revealed that the compound had no antibacterial activity against strains tested at concentrations <1024 µg/mL. The combination of the flavonoid at a concentration of 128 µg/mL with gentamicin presented synergistic effects against S. aureus 10 and E. coli 06, and also reduced the MIC from 16 µg/mL to 4 µg/mL and 8 µg/mL, respectively. Amikacin also showed synergistic effects against S. aureus 10 and E. coli 06. We also observed reduced MIC for both, from 128 µg/mL to 32 µg/mL; however, antagonism for P. aeruginosa increased the MIC from 16 µg/mL to 64 µg/mL. The combination of the flavonoid with the aminoglycosides may be an alternative to potentiate the expected results in treatment against S. aureus and E. coli, since their association leads to a synergistic effect, reducing the MIC of these drugs and decreasing the dose necessary for therapeutic success.

Efficacy of Synbiotics to Reduce Symptoms and Rectal Inflammatory Response in Acute Radiation Proctitis: A Randomized, Double-Blind, Placebo-Controlled Pilot Trial.

Purpose: Evaluate whether the daily intake of synbiotics improves symptoms and rectal/systemic inflammatory response in patients with radiation-induced acute proctitis.Methods and Materials: Twenty patients who underwent three-dimensional conformal radiotherapy for prostate cancer were randomized to intake either a synbiotic powder containing Lactobacillus reuteri (108 CFU) and soluble fiber (4.3 g) or placebo. EORTC QLQ-PRT23 questionnaire was applied before the beginning of radiotherapy and after the fifth, sixth, and seventh weeks of treatment, and the sum of both the complete (proctitis symptoms plus quality of life) and partial (proctitis symptoms) scores were compared. Fecal calprotectin was measured at Day 0 and in the fourth week of treatment, and serum C-reactive protein/albumin ratio were measured in the fourth week of treatment.Results: Both the complete and partial questionnaire score (median and range) were higher in the fifth and sixth weeks in the placebo group; there was a higher increase in fecal calprotectin in the placebo group and no difference comparing CRP/albumin ratio.Conclusions: Synbiotics reduce proctitis symptoms and improve quality of life by preventing rectal inflammation during radiotherapy for prostate cancer.

Chemical composition, antioxidant, antimicrobial and antibiofilm activities of Vitex gardneriana schauer leaves's essential oil.

This study aimed to determinate the chemical composition and evaluate the antimicrobial and antioxidant activity of the essential oil obtained from leaves of V. gardneriana. The Vitex gardneriana leaves's were hydrodistilled to obtain the essential oil and the chemical composition determined by GC/MS analysis. The antimicrobial activities were determined by microdilution method. The activity of essential oil on biofilm was evaluated by quantification of total biomass and enumeration of biofilm-entrapped viable cells. The antioxidant activity was assessed by DPPH free radical assay, ferrous ion chelating assay, ferric-reducing antioxidant power and ß-carotene bleaching assay. Furthermore, the essential oil was tested on viability of health human, animal cells and the microcrustacean Artemia sp. The essential oil showed high content of sesquiterpenes and very low content of monoterpenes. Regarding activity on planktonic cells, the essential oil reduced the growth of the all species tested but showed MIC values only to S. aureus (0.31%). In general, the essential oil reduced significantly the biofilm biomass and the number of viable cells of bacteria and yeasts, mainly on biofilm formation. The essential oil showed a potential antioxidant activity, mainly on ß-carotene oxidation. Moreover, the essential oil reduced the cell viability of murine fibroblasts but not show viability reduction of human keratinocytes. Furthermore, the oil not show toxicity against the microcrustacean. Thus, the essential oil from V. gardneriana leaves may be considered as an important alternative against biofilms formed by bacteria and yeasts related to infections, as well as a natural antioxidant and non-toxic substance on human cells.

Nitrogen maintenance requirements and potential for nitrogen retention of pullets in growth phase.

Experiments were conducted to estimate daily N maintenance requirements (NMRs) and the genetic potential for daily N retention (NRmax T) of pullets in growth phase. Three nitrogen balance trials were conducted, and a total of 48 Hy-line W-36 pullets were used in each trial in age periods (starter: 14-28, grower: 56-70 and developer: 98-112 days). The treatments consisted of six graded levels of nitrogen in the diets (L1 = 8, L2 = 16, L3 = 24, L4 = 32, L5 = 40 and L6 = 48 g N/kg of feed), formulated using the dilution technique. The regression analyses between nitrogen intake and nitrogen excretion were performed to fit the exponential function and to determine the NMR. The daily NMRs that were estimated at 294, 331 and 355 mg/BWkg 0.67 for the initial, grower and developer periods, respectively, were applied for further calculation of NRmax T as the threshold value of the function between N intake and daily N balance. The NRmax T was estimated by a statistical procedure following several iteration steps by the Levenberg-Marquardt algorithm until the sum of the squares of the residual was minimized. The NRmax T was estimated at 3,200, 2,633 and 1,826 mg/BWkg 0.67 for starter, grower and developer periods respectively. The determined model parameters were the precondition for modelling of the amino acid requirement based on an exponential N-utilization model and depended on performance and dietary amino acid efficiency. This procedure will be further developed and applied in the subsequent study.

Optimal in-feed amino acid ratio for laying hens based on deletion method.

A total of 56 Hy-line W-36 hens from 28 to 30 weeks were used on nitrogen balance (NB) trial to estimate daily N maintenance requirements (NMR) and the genetic potential for daily N retention (NRmax T). The treatments consisted of six graded levels of nitrogen in the diets (N1 = 8; N2 = 16; N3 = 24; N4 = 32; N5 = 40; and N6 = 48 g N/kg of feed), formulated using the dilution technique. The regression analyses between nitrogen intake and excretion were performed to fit the exponential function and to determine the NMR = 292 mg/BWkg 0.67 , which was applied for further calculation of NRmax T = 1,883 mg/BWkg 0.67 . A second NB trial was conducted, and a total of 96 Hy-line W-36 hens were used in the same period to estimate the ideal amino acid ratio (IAAR). Twelve treatments with eight replicates and one bird per cage were used. A balanced diet (BD) was formulated to meet the IAAR and the requirement of other nutrients for pullets. The limiting diets were formulated diluting BD with cornstarch and refilled with synthetic AAs and other feed ingredients, except for the AA under study. In each trial, the data of nitrogen intake, excretion, deposition and retention were obtained in a NB trial. The IAAR determined by Goettingen approach was Lys 100, Met+Cys 88, Trp 21, Thr 69, Arg 109, Val 90, Ile 75, Leu 127, Phe+Tir 110, Gly+Ser 73 and His 29%. The IAAR determined by Louvain approach was Lys 100, Met+Cys 88, Trp 21, Thr 69, Arg 104, Val 91, Ile 78, Leu 121, Phe+Tir 119, Gly+Ser 77 and His 29%.

Potentially inappropriate medications: predictor for mortality in a cohort of community-dwelling older adults.

PURPOSE: The objective of this study was to investigate whether the potentially inappropriate medication (PIM) use is a predictor for mortality in a community-based population of older adults. METHODS: A cohort study was performed between January 1, 1997, and December 31, 2011, based on data from a representative sample of the population aged 60 years or older living in Bambuí, Brazil. Univariate and multivariate analyses of the association between the variable of interest (use of PIM or pharmacological groups of PIM) and mortality were based on the extended Cox model for proportional hazards. The extended model was adopted to include the measurement of the exposure of interest throughout the follow-up period and not only at baseline. Adjustment variables included sociodemographic characteristics, health status, and use of health care services and medications. A level of significance of 5% was adopted for all analyses. RESULTS: The prevalence of PIM use was 56.0% (95%CI 53.4-58.6). After multiple adjustments, the risk of death among users of at least one PIM was 44% higher (HR = 1.44; 95%CI 1.21 to 1.71) than among those who did not use any PIM. Among the PIM groups analyzed, antipsychotics were the most strongly associated with mortality (HR = 2.33; 95%CI 1.72 to 3.17). CONCLUSIONS: The study revealed a high prevalence of PIM use among the community-based aged population. It also identified PIM use as a predictor for mortality in this group of individuals. This indicates the need for selection of safer drug therapy alternatives in this patient group.

Molecular identification of Histoplasma capsulatum using rolling circle amplification.

Histoplasmosis is a systemic fungal disease that occurs worldwide, causing symptomatic infection mostly in immunocompromised hosts. Etiological agent is the dimorphic fungus, Histoplasma capsulatum, which occurs in soil contaminated with bird or bat droppings. Major limitation in recognition of H. capsulatum infections is the low awareness, since other diseases may have similar symptomatology. The molecular methods have gained importance because of unambiguous diagnostic ability and efficiency. The aim of this study was to develop and evaluate a padlock probe in view of rolling circle amplification (RCA) detection method which targets ITS (Internal Transcribed Spacer) rDNA of H. capsulatum enabling rapid and specific detection of the fungus in clinical samples. Two padlock probes were designed and one of these (HcPL2) allowed specific amplification of H. capsulatum DNA while no cross-reactivity was observed with fungi used as negative controls. This method proved to be effective for H. capsulatum specific identification and demonstrated to be faster than the traditional method of microbiological identification.

Shared Physiological Traits of Exophiala Species in Cold-Blooded Vertebrates, as Opportunistic Black Yeasts.

Several species of the genus Exophiala are found as opportunistic pathogens on humans, while others cause infections in cold-blooded waterborne vertebrates. Opportunism of these fungi thus is likely to be multifactorial. Ecological traits [thermotolerance and pH tolerance, laccase activity, assimilation of mineral oil, and decolorization of Remazol Brilliant Blue R (RBBR)] were studied in a set of 40 strains of mesophilic Exophiala species focused on the salmonis-clade mainly containing waterborne species. Thermophilic species and waterborne species outside the salmonis-clade were included for comparison. Strains were able to tolerate a wide range of pHs, although optimal growth was observed between pH 4.0 and 5.5. All strains tested were laccase positive. Strains were able to grow in the presence of the compounds (mineral oil and RBBR) with some differences in assimilation patterns between strains tested and also were capable of degrading the main chromophore of RBBR. The study revealed that distantly related mesophilic species behave similarly, and no particular trend in evolutionary adaptation was observed.

Fonsecaea pugnacius, a Novel Agent of Disseminated Chromoblastomycosis.

We report a fatal case of a chromoblastomycosis-like infection caused by a novel species of Fonsecaea in a 52-year-old immunocompetent Caucasian male from an area of chromoblastomycosis endemicity in Brazil. The patient had a 30-year history of slowly evolving, verrucous lesions on the right upper arm which gradually affected the entire arm, the left hemifacial area, and the nose. Subsequent dissemination to the brain was observed, which led to death of the patient. The internal transcribed spacer (ITS) and partial large subunit (LSU), BT2, and CDC42 genes of the isolates recovered from skin and brain were sequenced, confirming the novelty of the species. The species is clinically unique in causing brain abscesses secondary to chromoblastomycosis lesions despite the apparent intact immunity of the patient. Histopathologic appearances were very different, showing muriform cells in skin and hyphae in brain.

Recombinant adenovirus as a model to evaluate the efficiency of free chlorine disinfection in filtered water samples.

BACKGROUND: In Brazil, ordinance no. 2,914/2011 of the Ministry of Health requires the absence of total coliforms and Escherichia coli (E. coli) in treated water. However it is essential that water treatment is effective against all pathogens. Disinfection in Water Treatment Plants (WTP) is commonly performed with chlorine. METHODS: The recombinant adenovirus (rAdV), which expresses green fluorescent protein (GFP) when cultivated in HEK 293A cells, was chosen as a model to evaluate the efficiency of chlorine for human adenovirus (HAdV) inactivation in filtered water samples from two WTPs: Lagoa do Peri (pH 6.9) and Morro dos Quadros (pH 6.5). Buffered demand free (BDF) water (pH 6.9 and 8.0) was used as control. The samples were previously submitted to physicochemical characterization, and bacteriological analysis. Two free chlorine concentrations and two temperatures were assayed for all samples (0.2 mg/L, 0.5 mg/L, and 15°C, and 20°C). Fluorescence microscopy (FM) was used to check viral infectivity in vitro and qPCR as a molecular method to determine viral genome copies. Real treated water samples from the WTP (at the output of WTP and the distribution network) were also evaluated for total coliforms, E. coli and HAdV. RESULTS: The time required to inactivate 4log10 of rAdV was less than 1 min, when analyzed by FM, except for BDF pH 8.0 (up to 2.5 min for 4log10). The pH had a significant influence on the efficiency of disinfection. The qPCR assay was not able to provide information regarding rAdV inactivation. The data were modeled (Chick-Watson), and the observed Ct values were comparable with the values reported in the literature and smaller than the values recommended by the EPA. In the treated water samples, HAdV was detected in the distribution network of the WTP Morro dos Quadros (2.75 × 10(3) PFU/L). CONCLUSION: The Chick-Watson model proved to have adjusted well to the experimental conditions used, and it was possible to prove that the adenoviruses were rapidly inactivated in the surface water treated with chlorine and that the recombinant adenovirus expressing GFP is a good model for this evaluation.

Medication Literacy Test for Older Adults: psychometric analysis and standardization of the new instrument.

BACKGROUND: Low medication literacy is prevalent among older adults and is associated with adverse drug events. The Medication Literacy Test for Older Adults (TELUMI) was developed and content validated in a previously published study. AIM: To evaluate the psychometric properties and provide norms for TELUMI scores. METHOD: This was a cross-sectional methodological study with older adults selected from the community and from two outpatient services. Descriptive item-analysis, exploratory factor analysis (EFA), item response theory (IRT), reliability, and validity analysis with schooling and health literacy were performed to test the psychometric properties of the TELUMI. The classification of the TELUMI scores was performed using percentile norms. RESULTS: A total of 344 participants, with a mean age of 68.7 years (standard deviation = 6.7), were included; most were female (66.6%), black/brown (61.8%), had low schooling level (60.2%) and low income (55.2%). The EFA pointed to the one-dimensional structure of TELUMI. A three-parameter logistic model was adopted for IRT. All items had an adequate difficulty index. One item had discrimination < 0.65, and three items had an unacceptable guessing index (< 0.35) and were excluded. The 29-item version of TELUMI had excellent internal consistency (KR20 = 0.89). There was a positive and strong association between TELUMI scores and health literacy and education level. The scores were classified as inadequate medication literacy (≤ 10.0 points), medium medication literacy (11-20 points), and adequate medication literacy (≥ 21 points). CONCLUSION: The results suggest that the 29-item version of TELUMI is psychometrically adequate for measuring medication literacy in older adults.

Assessment of oxidative stress biomarkers in the threatened annual killifish Austrolebias charrua exposed to Roundup.

This study aimed to analyze the toxic effects of Roundup Transorb® on the endangered Neotropical annual killifish Austrolebias charrua through the assessment of molecular and biochemical biomarkers. The fish were collected in temporary ponds and exposed to environmentally realistic concentrations of the herbicide (5 mg.L-1 for 96 h). The production of ROS, lipid peroxidation, DNA damage, and membrane fluidity were evaluated in the blood cells by flow cytometry. The mRNA expression of the antioxidant-related genes sod2, cat, gstα, atp1a1, gclc, and ucp1 across the brain, liver, and gills was quantified. The acute exposure of annual killifish to Roundup significantly increased ROS production, lipid peroxidation, and DNA damage in their erythrocytes. Likewise, Roundup Transorb® decreased membrane fluidity in the blood cells of the exposed fish. Gene expression analysis revealed that Roundup exposure alters the relative expression of genes associated with oxidative stress and antioxidant defense. Our results give rise to new insights into adaptive mechanisms of A. charrua in response to Roundup. Since Brazilian annual killifishes strongly risk extinction, this study paves the way for developing novel biotechnologies applied to environmental monitoring and aquatic toxicology assessment.