Analysis of DNA methylation markers for tissue identification in individuals with different clinical phenotypes.

Deoxyribonucleic acid (DNA) methylation patterns can be used to identify the type of tissue or body fluid found at a crime scene. However, tissue-related methylation levels have not been analyzed in individuals with different illnesses and medical conditions in forensic-specific studies. The primary goal of this study was to investigate if certain clinical phenotypes can alter the methylation levels of CpG sites in genes involved in tissue typing. Four studies with focus on DNA methylation analysis on individuals with different clinical conditions were selected from the Gene Expression Omnibus database. Then, a list of 137 CpG sites was compiled for further investigation. Statistical tests were performed to compare the beta-values results obtained for the control groups and the individuals affected by medical conditions. For each study, CpG sites that presented significant statistical differences between patients and control group were identified and it was possible to notice that DNA methylation levels can be affected in sites with potential forensic use. Although the observed DNA methylation variation (less than 10% difference) in this study would likely not cause any issues in body fluid identification, the results are important to show that this type of analysis should be taken into consideration when investigating and further validating body fluid markers. The CpG sites identified in this study should be further investigated by future studies on body fluids identification, and due to the significant difference in methylation levels in samples from affected individuals, caution must be taken before including these sites in tissue identification investigations.

Parents' Willingness to Vaccinate Children for COVID-19: Conspiracy Theories, Information Sources, and Perceived Responsibility.

Understanding parental decision-making about vaccinating their children for COVID-19 is essential to promoting uptake. We conducted an online survey between April 23-May 3, 2021, among a national sample of U.S. adults to assess parental willingness to vaccinate their child(ren). We also examined associations between parental intentions to VACCINATE their children for COVID-19 and conspiracy theory beliefs, trusted information sources, trust in public authorities, and perceptions regarding the responsibility to be vaccinated. Of 257 parents of children under 18 years that responded, 48.2% reported that they would vaccinate their children, 25.7% were unsure, and 26.1% said they would not vaccinate. After adjusting for covariates, each one-point increase in the Vaccine Conspiracy Beliefs Scale was associated with 25% lower odds of parents intending to vaccinate their children compared to those who did not intend to (adjusted odds ratio (AOR) = 0.75, 95% confidence interval (CI): 0.64-0.88). Parents that perceived an individual and societal responsibility to be vaccinated were more likely to report that they intended to vaccinate their children compared to those that did not intend to vaccinate their children (AOR = 5.65, 95% CI: 2.37-13.44). Findings suggest that interventions should focus on combatting conspiracy beliefs, promoting accurate and trusted information sources, and creating social norms emphasizing shared responsibility for vaccination.

Detection of sensitivity and vestigiality of presumptive tests for swabbed blood stains.

It is a common practice in forensic casework to use presumptive tests for blood stains before DNA extraction and testing. Stains are usually swabbed and then the swabs are sent for analysis. The Kastle-Meyer (KM) and Leucomalachite green (LMG) presumptive tests for blood are widely used, and their sensitivities have been thoroughly tested in the literature in solution and directly on stains, but not on swabbed stains to mimic casework. In this study, the sensitivity of the KM and LMG tests was tested on eight blood dilutions on cotton fabric and ceramic tile that were stained and subsequently swabbed. Both tests showed sensitivity up to 1:5000, which is slightly lower than reported values in solution or directly on stain but still highly effective in most cases. Stains were also cleaned with common agents, then swabbed and re-tested. Stained ceramic tiles cleaned with soap/water or bleach gave mixed positive and negative results for the 1:10 dilution, presumably due to variance in how thoroughly each investigator cleaned the stain, and other dilutions were undetectable after cleaning. The LMG test gave false positives for bleach cleaned stains, due to reagent reactivity with bleach. Surprisingly, blood was detectible up to the 1:100 dilution with both tests on stained cotton fabric that was cleaned in a washing machine with detergent and dried. Ultimately the KM and LMG presumptive tests remain effective tools for swabbed blood stains, and their practicality for cleaned stains is dependent on material containing the stain, cleaning agent and processing.

High frequency of increased triclosan MIC among CC5 MRSA and risk of misclassification of the SCCmec into types.

BACKGROUND: Typing of staphylococcal cassette chromosome mec (SCCmec) elements is commonly used for studies on the molecular epidemiology of MRSA. OBJECTIVES: To perform an investigation centred on uncovering the reasons for misclassification of MRSA clonal complex 5 (CC5) SCCmec type II clinical isolates in our laboratory. METHODS: MRSA isolates from CC5 were subjected to WGS and SCCmec typing. RESULTS: This investigation led to the discovery that the classification failure was due to an insertion of IS1272 carrying the fabI gene on a transposable element (TnSha1) that confers increased MIC to the biocide triclosan. Genomic analysis revealed that fabI was present in 25% of the CC5 MRSA isolates sampled. The frequency of TnSha1 in our collection was much higher than that observed among publicly available genomes (0.8%; n = 24/3142 CC5 genomes). Phylogenetic analyses revealed that genomes in different CC5 clades carry TnSha1 inserted in different integration sites, suggesting that this transposon has entered CC5 MRSA genomes on multiple occasions. In at least two genotypes, ST5-SCCmecII-t539 and ST5-SCCmecII-t2666, TnSha1 seems to have entered prior to their divergence. CONCLUSIONS: Our work highlights an important misclassification problem of SCCmecII in isolates harbouring TnSha1 when Boye's method is used for typing, which could have important implications for molecular epidemiology of MRSA. The importance of increased-MIC phenotype is still a matter of controversy that deserves more study given the widespread use of triclosan in many countries. Our results suggest expanding prevalence that may indicate strong selection for this phenotype.

Intrauterine and Lactational Exposure to Paracetamol: Cardiometabolic Evaluation in Adult Female and Male Offspring.

ABSTRACT: Paracetamol (PAR) is the most common over-the-counter drug recommended by physicians for treatment of pain and fever during gestation. This drug is not teratogenic, being considered safe for fetus; however, PAR crosses the blood-placental barrier. Considering that, the present study aimed to evaluate the vascular and metabolic safety of PAR exposure during intrauterine and neonatal development in adult male and female-exposed offspring. Wistar female rats were gavaged, with PAR (350 mg/kg/d), from gestational day 6-21 or from gestational day 6 until postnatal day 21. Control dams received water by gavage at the same periods. The male and female offspring were evaluated at adulthood (80 days of life). The thoracic aorta reactivity to acetylcholine, sodium nitroprusside, and phenylephrine was evaluated in male and female adult offspring. It was observed that aortic relaxation was similar between the PAR and control offspring. In addition, the contraction to phenylephrine was similar between the groups. Further, the insulin sensitivity, adipose tissue deposition and blood pressure were not different between PAR and control adult offspring. These results suggest that the protocol of PAR exposure used in the present study did not program vascular and metabolic alterations that would contribute to the development of cardiometabolic diseases in adult life, being safe for the exposed offspring.

Anthelmintic resistance of gastrointestinal nematodes in sheep grazing in irrigated and dry areas in the semiarid region of northeastern Brazil.

This study aimed to determine the AR of gastrointestinal nematodes (GIN) to commercial drugs in sheep flocks naturally infected, grazing in irrigated (IA) and dry (DA) areas of the semiarid region in northeastern Brazil. Fecal egg count reduction tests (FECRT) were performed at 10 farms. From each flock, 36 adult sheep were selected and divided into five groups (G1 (0.08% ivermectin), G2 (10% albendazole), G3 (5% levamisole), G4 (1% moxidectin), G5 (10% closantel) and one control group, G6). All the commercial drugs were found to reduce the number of eggs per gram of feces (EPG). Resistance to ivermectin (37.1%), albendazole (52.1%), and levamisole (52.0%) was detected at all the farms, but nematodes proved to be susceptible to moxidectin (87.9%) and closantel (83.9%). The overall average efficacy of the commercial drugs was significantly higher (P < 0.05) in DA (49.2%), where moxidectin (90.4%) showed high effectiveness. The presence of the parasite Haemonchus contortus predominated at all the farms. The variables irrigated area (P = 0.002), intensive breeding (P = 0.018), uncovered enclosures (P = 0.05), cultivated (P = 0.043) and native/cultivated (P = 0.007) pastures, and rotational grazing (P = 0.013) were significantly associated with GIN infection; irrigated area (P = 0.009), semi-intensive breeding (P = 0.05), rotational grazing (P = 0.045), cultivated (P = 0.021) and native/cultivated (P = 0.04) pastures, and estimated weighing of animals (P = 0.002) were significantly associated with AR. Therefore, improved management practices and strategic deworming must be implemented to prevent the development of AR.

Paternity testing using massively parallel sequencing and the PowerSeq™ AUTO/Y system for short tandem repeat sequencing.

Massively parallel sequencing (MPS) is gaining attention as a new technology for routine forensic casework, including paternity testing. Recently released MPS multiplex panels provide many more loci compared to CE methods, plus provide sequence-based alleles that together improve the statistical power of the genetic testing. Here, an MPS system (PowerSeq™ AUTO/Y) was applied for STR sequencing in the study of first-degree STR sequence allele inheritance from families in Southern Brazil. In 29 trios (mother-child-father) analyzed, the paternity index values generally increased when data from sequence-based analysis were used in comparison to length-based data. Further, allele inconsistencies (e.g., single repeat mutation events) between child and parents could be resolved with MPS by assessing the core repeat and flanking region sequences. Lastly, the sequence information allowed for identification of isoalleles (alleles of the same size, but different sequence) to determine specific paternal and maternal inheritances. The results from this study showed advantages of implementing sequence-based analysis, MPS, in paternity testing with improved statistical calculations and a greater resolution for the trios/families tested.

Forensic discrimination of vaginal epithelia by DNA methylation analysis through pyrosequencing.

The accurate identification of body fluids from crime scenes can aid in the discrimination between criminal and innocent intent. This research aimed to determine if the levels of DNA methylation in the locus PFN3A could be used to discriminate vaginal epithelia from other body fluids. In this work we bisulfite-modified and amplified DNA samples from blood, saliva, semen, and vaginal epithelia using primers for PFN3A. Through pyrosequencing we were able to show that vaginal epithelia present distinct methylation levels when compared to other body fluids. Mixtures of different body fluids present methylation values that correlate with single-source body fluid samples and the primers for PFN3A are specific for primates. This report successfully demonstrated that the analysis of methylation in the PFN3A locus can be used for vaginal epithelia discrimination in forensic samples.

High-resolution melt analysis of DNA methylation to discriminate semen in biological stains.

The goal of this study was to develop a method for the detection of semen in biological stains using high-resolution melt (HRM) analysis and DNA methylation. To perform this task, we used an epigenetic locus that targets a tissue-specific differentially methylated region for semen. This specific locus, ZC3H12D, contains methylated CpG sites that are hypomethylated in semen and hypermethylated in blood and saliva. Using this procedure, DNA from forensic stains can be isolated, processed using bisulfite-modified polymerase chain reaction (PCR), and detected by real-time PCR with HRM capability. The method described in this article is robust; we were able to obtain results from samples with as little as 1 ng of genomic DNA. Samples inhibited by humic acid still produced reliable results. Furthermore, the procedure is specific and will not amplify non-bisulfite-modified DNA. Because this process can be performed using real-time PCR and is quantitative, it fits nicely within the workflow of current forensic DNA laboratories. As a result, it should prove to be a useful technique for processing trace evidence samples for serological analysis.

Differential proteomic analysis of outer membrane enriched extracts of Bacteroides fragilis grown under bile salts stress.

Bacteroides fragilis is the most commonly isolated anaerobic bacteria from infectious processes. Several virulence traits contribute to the pathogenic nature of this bacterium, including the ability to tolerate the high concentrations of bile found in the gastrointestinal tract (GIT). The activity of bile salts is similar to detergents and may lead to membrane permeabilization and cell death. Modulation of outer membrane proteins (OMPs) is considered a crucial event to bile salts resistance. The primary objective of the current work was to identify B. fragilis proteins associated with the stress induced by high concentration of bile salts. The outer membrane of B. fragilis strain 638R was isolated after growth either in the presence of 2% conjugated bile salts or without bile salts. The membrane fractions were separated on SDS-PAGE and analyzed by ESI-Q/TOF tandem mass spectrometry. A total of 37 proteins were identified; among them nine were found to be expressed exclusively in the absence of bile salts whereas eight proteins were expressed only in the presence of bile salts. These proteins are related to cellular functions such as transport through membrane, nutrient uptake, and protein-protein interactions. This study demonstrates the alteration of OMPs composition in B. fragilis during bile salts stress resistance and adaptation to environmental changes. Proteomics of OMPs was also shown to be a useful approach in the identification of new targets for functional analyses.

The role of physical habitat and sampling effort on estimates of benthic macroinvertebrate taxonomic richness at basin and site scales.

Taxonomic richness is one of the most important measures of biological diversity in ecological studies, including those with stream macroinvertebrates. However, it is impractical to measure the true richness of any site directly by sampling. Our objective was to evaluate the effect of sampling effort on estimates of macroinvertebrate family and Ephemeroptera, Plecoptera, and Trichoptera (EPT) genera richness at two scales: basin and stream site. In addition, we tried to determine which environmental factors at the site scale most influenced the amount of sampling effort needed. We sampled 39 sites in the Cerrado biome (neotropical savanna). In each site, we obtained 11 equidistant samples of the benthic assemblage and multiple physical habitat measurements. The observed basin-scale richness achieved a consistent estimation from Chao 1, Jack 1, and Jack 2 richness estimators. However, at the site scale, there was a constant increase in the observed number of taxa with increased number of samples. Models that best explained the slope of site-scale sampling curves (representing the necessity of greater sampling effort) included metrics that describe habitat heterogeneity, habitat structure, anthropogenic disturbance, and water quality, for both macroinvertebrate family and EPT genera richness. Our results demonstrate the importance of considering basin- and site-scale sampling effort in ecological surveys and that taxa accumulation curves and richness estimators are good tools for assessing sampling efficiency. The physical habitat explained a significant amount of the sampling effort needed. Therefore, future studies should explore the possible implications of physical habitat characteristics when developing sampling objectives, study designs, and calculating the needed sampling effort.

Midwifery and quality care: findings from a new evidence-informed framework for maternal and newborn care.

In this first paper in a series of four papers on midwifery, we aimed to examine, comprehensively and systematically, the contribution midwifery can make to the quality of care of women and infants globally, and the role of midwives and others in providing midwifery care. Drawing on international definitions and current practice, we mapped the scope of midwifery. We then developed a framework for quality maternal and newborn care using a mixed-methods approach including synthesis of findings from systematic reviews of women's views and experiences, effective practices, and maternal and newborn care providers. The framework differentiates between what care is provided and how and by whom it is provided, and describes the care and services that childbearing women and newborn infants need in all settings. We identified more than 50 short-term, medium-term, and long-term outcomes that could be improved by care within the scope of midwifery; reduced maternal and neonatal mortality and morbidity, reduced stillbirth and preterm birth, decreased number of unnecessary interventions, and improved psychosocial and public health outcomes. Midwifery was associated with more efficient use of resources and improved outcomes when provided by midwives who were educated, trained, licensed, and regulated. Our findings support a system-level shift from maternal and newborn care focused on identification and treatment of pathology for the minority to skilled care for all. This change includes preventive and supportive care that works to strengthen women's capabilities in the context of respectful relationships, is tailored to their needs, focuses on promotion of normal reproductive processes, and in which first-line management of complications and accessible emergency treatment are provided when needed. Midwifery is pivotal to this approach, which requires effective interdisciplinary teamwork and integration across facility and community settings. Future planning for maternal and newborn care systems can benefit from using the quality framework in planning workforce development and resource allocation.

Evaluation of DNA methylation markers and their potential to predict human aging.

We present epigenetic methylation data for two genetic loci, GRIA2, and NPTX2, which were tested for prediction of age from different donors of biofluids. We analyzed 44 saliva samples and 23 blood samples from volunteers with ages ranging from 5 to 72 years. DNA was extracted and bisulfite modified using commercial kits. Specific primers were used for amplification and methylation profiles were determined by pyrosequencing. Methylation data from both markers and their relationship with age were determined using linear regression analysis, which indicates a positive correlation between methylation and age. Older individuals tend to have increased methylation in both markers compared to younger individuals and this trend was more pronounced in the GRIA2 locus when compared to NPTX2. The epigenetic predicted age, calculated using a GRIA2 regression analysis model, was strongly correlated to chronological age (R(2) = 0.801), with an average difference of 6.9 years between estimated and observed ages. When using a NPTX2 regression model, we observed a lower correlation between predicted and chronological age (R(2) = 0.654), with an average difference of 9.2 years. These data indicate these loci can be used as a novel tool for age prediction with potential applications in many areas, including clinical and forensic investigations.

Visually determined stream mesohabitats influence benthic macroinvertebrate assessments in headwater streams.

Mesohabitat components such as substrate and surface flow types are intimately related to benthic macroinvertebrate assemblages in streams. Visual assessments of the distribution of these components provide a means of evaluating physical habitat heterogeneity and aid biodiversity surveys and monitoring. We determined the degree to which stream site and visually assessed mesohabitat variables explain variability (i.e., beta-diversity) in the relative abundance and presence-absence of all macroinvertebrate families and of Ephemeroptera, Plecoptera, and Trichoptera (EPT) genera. We systematically sampled a wide variety of mesohabitat arrangements as they occured in stream sites. We also estimated how much of the explanation given by mesohabitat was associated with substrate or surface flow types. We performed variation partitioning to determine fractions of explained variance through use of partial redundancy analysis (pRDA). Mesohabitats and stream sites explained together from 23 to 32% of the variation in the four analyses. Stream site explained 8-11% of that variation, and mesohabitat variables explained 13-20%. Surface flow types accounted for >60% of the variation provided by the mesohabitat component. These patterns are in accordance with those obtained in previous studies that showed the predominance of environmental variables over spatial location in explaining macroinvertebrate distribution. We conclude that visually assessed mesohabitat components are important predictors of assemblage composition, explaining significant amounts of beta-diversity. Therefore, they are critical to consider in ecological and biodiversity assessments involving macroinvertebrates.

[Integrative review: concepts and methods used in nursing]. / Revisão integrativa: conceitos e métodos utilizados na enfermagem.

UNLABELLED: Integrative review (IR) has an international reputation in nursing research and evidence-based practice. This IR aimed at identifying and analyzing the concepts and methods recommended to undertaking IR in nursing. Nine information resources,including electronic databases and grey literature were searched. Seventeen studies were included. The results indicate that: primary studies were mostly from USA; it is possible to have several research questions or hypotheses and include primary studies in the review from different theoretical and methodological approaches; it is a type of review that can go beyond the analysis and synthesis of findings from primary studies allowing exploiting other research dimensions, and that presents potentialities for the development of new theories and new problems for research. CONCLUSION: IR is understood as a very complex type of review and it is expected to be developed using standardized and systematic methods to ensure the required rigor of scientific research and therefore the legitimacy of the established evidence.

Immediate effects of photobiomodulation on saliva production. / Efeitos imediatos da fotobiomodulação na produção de saliva.

PURPOSE: To verify the immediate effects of photobiomodulation on the production of salivary flow and the correlation of demographic, anthropometric and medication use data. METHODS: The study included 100 healthy individuals, aged between 18 and 76 years (mean 27.2 years), randomly split into an experimental group and a placebo group. Assessments of anthropometric measurements, self-perception of saliva production and sialometry were performed. Next, LASER irradiation was carried out at an infrared wavelength (808 nanometers) with 100 milliwatts (mw) of power at five intraoral points: on the sublingual glands and bilaterally on the submandibular and parotid glands, at doses of 9, 18 and 24 joules (J). Sialometry was repeated after each application. The control group received the same procedures with placebo equipment. RESULTS: There was a statistical association in the self-perception of reduced saliva in the experimental group for the 24J dose and in sialometry and in the reduction in salivary flow for the 18J and 24J doses and an increase to 9J, in both groups. There was no association when comparing the experimental and placebo groups. Multiple multinomial regression analysis revealed that the reduction or increase in salivary flow is independent of demographic, anthropometric and medication use variables. CONCLUSION: The bioinhibitory action of photobiomodulation on healthy salivary glands occurred at a dose of 18J and 24J, while the biostimulant action happened at a dose of 9J, regardless of demographic, anthropometric variables and medication use. The self-perception of reduced salivary flow occurred at 24J.

OBJETIVO: Verificar os efeitos imediatos da fotobiomodulação na produção do fluxo salivar e a correlação dos dados demográficos, antropométricos e de uso de medicamentos. MÉTODO: Participaram do estudo 100 indivíduos saudáveis, com idade entre 18 e 76 anos (média 27,2 anos), divididos de forma randomizada em grupo experimental e grupo placebo. Foram realizadas as avaliações das medidas antropométricas, autopercepção da produção de saliva e a sialometria. Na sequência, realizou-se a irradiação do LASER no comprimento de onda infravermelho (808 nanômetros) com 100 miliwatts (mw) de potência em cinco pontos intraorais: nas glândulas sublingual e bilateralmente nas submandibulares e parótidas, nas doses 9, 18 e 24 joules (J). A sialometria foi repetida após cada aplicação. O grupo controle recebeu os mesmos procedimentos com equipamento placebo. RESULTADOS: Houve associação estatística na autopercepção de redução da saliva no grupo experimental para a dose de 24J e na sialometria e na redução do fluxo salivar para as doses 18J e 24J e aumento para 9J, em ambos os grupos. Não houve associação quando comparado entre os grupos experimental e placebo. A análise de regressão multinomial múltipla revelou que a redução ou o aumento do fluxo salivar independe das variáveis demográficas, antropométricas e uso de medicamentos. CONCLUSÃO: A ação bioinibitória da fotobiomodulação sobre as glândulas salivares saudáveis ocorreu em dose de 18J e 24J, já ação bioestimulante na dose 9J, independe das variáveis demográficas, antropométricas e uso de medicamentos. A autopercepção da redução do fluxo salivar ocorreu em 24J.

Topiramate treatment during adolescence induces short and long-term alterations in the reproductive system of female rats.

Topiramate (TPM) is an antiepileptic drug used for treating epilepsy in children, and migraine in teenagers. In this context, preclinical studies with adult female rats observed reproductive system abnormalities following treatment with TPM. Additionally, exposure to endocrine disruptors during developmental plasticity periods, such as childhood and adolescence, may influence characteristics in the adult individual. This study evaluated whether treatment with TPM during developmental periods influences the reproductive system of female rats either immediately or in adult life. Female Wistar rats were treated with TPM (41 mg/Kg/day) by oral gavage from postnatal day (PND) 16-28, or PND 28-50, which correspond to childhood and adolescence, respectively, and euthanized either 24 h after the final administration or during adulthood. Treatment with TPM during adolescence induced short-term increase in uterus and ovary weights and reduction in endometrial stroma thickness. Adult animals treated during adolescence displayed reduced primordial ovarian follicles' numbers, and increased primary and pre-antral ovarian follicles' numbers. Treatment during childhood induced no short or long-term differences. These results indicate TPM treatment during adolescence is capable of inducing short and long-term alterations on the reproductive system of female Wistar rats.

Vascular dysfunction programmed in male rats by topiramate during peripubertal period.

AIM: The present study evaluated whether topiramate (TPM) treatment during the peripubertal period affects vascular parameters of male rats and whether oxidative stress plays a role in these changes. MAIN METHODS: Rats were treated with TPM (41 mg/kg/day, gavage) or vehicle (CTR group) from the postnatal day (PND) 28 to 50. At PND 51 and 120 the rats were evaluated for: thoracic aorta reactivity to phenylephrine, in the presence (Endo+) or absence of endothelium (Endo-), to acetylcholine and to sodium nitroprusside (SNP), aortic thickness and endothelial nitric oxide synthase (eNOS) expression. In serum were analyzed: the antioxidant capacity by ferric reducing antioxidant power assay; endogenous antioxidant reduced glutathione, and superoxide anion. Results were expressed as mean ± s.e.m., differences when p < 0.05. STATISTICS: Two-way ANOVA (and Tukey's) or Student t-test. KEY FINDINGS: At PND 51, the contraction induced by phenylephrine in Endo+ ring was higher in TPM when compared to CTR. At PND 120, the aortic sensitivity to acetylcholine in TPM rats was reduced in comparison with CTR. The aortic eNOs expression and the aortic thickness were similar between the groups. At PND 51 and 120, TPM group presented a decrease in antioxidants when compared to CTR groups and at PND 120, in TPM group the superoxide anion was increased. SIGNIFICANCE: Taken together, the treatment of rats with TPM during peripubertal period promoted permanent impairment of endothelial function probably mediated by oxidative stress.