Exposure to Pesticides and Breast Cancer in an Agricultural Region in Brazil.

Rural workers are disproportionally exposed to pesticides and might be at an increased risk of developing chronic diseases. Here, we investigated the impact of pesticide exposure on breast cancer (BC) risk and disease profile in rural female workers. This is a case-control study that prospectively included 758 individuals. The study was conducted in the Southwest region of Paraná state in Brazil, a region characterized by family-based agriculture and intensive use of pesticides. We found that this region has a 41% higher BC diagnosis rate and 14% higher BC mortality rate than the mean rates in Brazil, as well as a pesticide trade volume about 6 times higher than the national average. We showed substantial exposure in this population and found that even women who did not work in the fields but performed equipment decontamination and clothes washing of male partners who worked in the fields had urine samples positive for glyphosate, atrazine, and/or 2,4-D. The crude association showed a significantly higher risk of BC among women exposed to pesticides (OR: 1.58, 95% CI 1.18-2.13). Adjusted analyses showed a lower and nonstatistically significant association (OR: 1.30, 95% CI 41 0.87-1.95). Stratification on disease profile showed a significantly higher risk of lymph node metastasis (adjusted OR: 2.19, 95% CI 1.31-3.72) in women exposed to pesticides. Our findings suggest that female populations exposed to pesticides are at a higher risk of developing BC with a more aggressive profile and draw attention to the need to monitor rural populations potentially exposed to pesticides in the field or at home.

The implications of DNA methylation for amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a complex and serious neurodegenerative disorder that develops in consequence of the progressive loss of the upper and lower motor neurons. Cases of ALS are classified as sporadic (sALS), or familial (fALS). Over 90% of cases are sALS, while roughly 10% are related to inherited genetic mutations (fALS). Approximately 70% of the genetic mutations that contribute to fALS have been identified. On the other hand, the majority of the sALS cases have an undetermined genetic contributor and few mutations have been described, despite the advanced genetic analysis methods. Also, several factors contribute to the onset and progression of ALS. Numerous lines of evidence indicate that epigenetic changes are linked to aging, as well as neurodegenerative disorders, such as ALS. In most cases, they act as the heritable regulation of transcription by DNA methylation, histone modification and expression of noncoding RNAs. Mechanisms involving aberrant DNA methylation could be relevant to human ALS pathobiology and therapeutic targeting. Despite advances in research to find factors associated with ALS and more effective treatments, this disease remains complex and has low patient survival. Here, we provide a narrative review of the role of DNA methylation for this complex neurodegenerative disorder.

Widespread pesticide contamination of drinking water and impact on cancer risk in Brazil.

Pesticides, which are associated with endocrine dysfunction, immunological dysregulation, and cancer, are widespread sources of drinking water contamination. The state of Paraná has a population of 11 million, is the second largest grain producer in Brazil and is a leading consumer of pesticides. In this study, we analyzed the extent of drinking water contamination from 11 proven, probable, or potentially carcinogenic pesticides (alachlor, aldrin-dieldrin, atrazine, chlordane, DDT-DDD-DDE, diuron, glyphosate-AMPA, lindane-γ-HCH, mancozeb-ETU, molinate, and trifluralin) in 127 grain-producing municipalities in the state of Paraná. Extensive contamination of drinking water was found, including legacy pesticides such as aldrin-dieldrin (mean 0.047 ppb), DDT-DDD-DDE (mean: 0.07), chlordane (mean: 0.181), and lindane-HCH (mean: 2.17). Most of the municipalities were significantly above the maximum limits for each one of the currently allowed pesticides (67% for alachlor, 9.44% for atrazine, 96.85% for diuron, 100% for glyphosate-AMPA, 80.31% for mancozeb-ETU, 91.33% for molinate, and 12.6% for trifluralin). Ninety-seven percent of municipalities presented a sum of all pesticides at levels significantly above (189.84 ppb) the European Union preconized limits (<0.5 ppb). Using the mean pesticide concentration in water (ppb), the exposed population for each municipality, and the benchmark cancer risk for pesticides, we estimated the minimum number of cancer cases attributable to pesticide-contaminated drinking water during the period (total of 542 cases). More than 80% were attributed to mancozeb-ETU and diuron. Glyphosate-AMPA and diuron-attributable cases strongly correlated with the total cancer cases in the same period (R = 0.8117 and 0.8138, respectively) as well as with breast cancer cases (R = 0.7695 and 0.7551, respectively). Water contamination was significantly correlated with the sum of the estimated cancer cases for all 11 pesticides detected in each city (R = 0.58 and p < 0.0001). These findings reveal extensive contamination of drinking water in the state of Paraná and suggest that contamination may increase the risk of cancer in this region.

The role of H3K9 acetylation and gene expression in different brain regions of Alzheimer's disease patients.

Aims: To evaluate H3K9 acetylation and gene expression profiles in three brain regions of Alzheimer's disease (AD) patients and elderly controls, and to identify AD region-specific abnormalities. Methods: Brain samples of auditory cortex, hippocampus and cerebellum from AD patients and controls underwent chromatin immunoprecipitation sequencing, RNA sequencing and network analyses. Results: We found a hyperacetylation of AD cerebellum and a slight hypoacetylation of AD hippocampus. The transcriptome revealed differentially expressed genes in the hippocampus and auditory cortex. Network analysis revealed Rho GTPase-mediated mechanisms. Conclusions: These findings suggest that some crucial mechanisms, such as Rho GTPase activity and cytoskeletal organization, are differentially dysregulated in brain regions of AD patients at the epigenetic and transcriptomic levels, and might contribute toward future research on AD pathogenesis.

Alzheimer's disease (AD) is the most common form of dementia affecting the elderly population. The onset and progression of AD are influenced by environmental factors, which are able to promote epigenetic changes on the DNA and/or the DNA-associated proteins called histones. We investigated a specific epigenetic modification of histones (H3K9 acetylation) in three brain regions of AD patients and compared them with elderly controls. We found increased levels of H3K9 acetylation in the cerebellum of AD patients, as well as a slight decrease of this modification in the hippocampus of the same patients. These brain tissues from AD patients showed abnormal gene expression patterns when compared with elderly controls. These findings contribute to understanding the molecular changes that occur in AD, and provide a basis for future research or drug development for AD treatment.

Effect of the bileaflet inlet valve angle on the flow of a pediatric ventricular assist device: Experimental analysis.

BACKGROUND: Mechanical heart valves (MHV) and its fluid dynamics inside a pulsatile pediatric ventricular assist device (PVAD) can be associated with blood degradation. In this article, flow structures are analyzed and compared by an experimental investigation on the effect of bileaflet MHV positioned at varying angles in the inlet port orifice of a PVAD. METHODS: Time-resolved particle image velocimetry was applied to characterize the internal flow of the device. St Jude Medical bileaftlet valves were used on the inlet orifice and positioned at 0°, 15°, 30°, 45°, 60°, and 90° in relation to the centerline of the device. Three planes with bidimensional velocity magnitude fields were considered in the analysis with visualization of diastolic jets, device wall washing patterns and flow circulation during emptying or systole of the pump. Also, the washing vortex area, and vertical velocity probabilities of regurgitant flows in the inlet valve were evaluated. RESULTS: The results show that a variation in the angle of the MHV at the inlet port produced distinct velocities, fluid structures, and regurgitant flow probabilities within the device. MHV positioned at an angle of 0° generated the strongest inlet jet, larger vortex area during filling, more prominent outgoing flow, and less regurgitation compared to the angles studied. The presence of unfavorable fluid structures, such as small vortices, and/or sudden flow structure interruption, and/or regurgitation, were identified at 45° and 90° angles. CONCLUSIONS: The 0° inlet angle had better outcomes than other angles due to its consistency in the multiple parameters analyzed.

Post-transcriptional diversity in riboproteins and RNAs in aging and cancer.

Post-transcriptional (PtscM) and post-translational (PtrnM) modifications of nucleotides and amino acids are covalent modifications able to change physio-chemical properties of RNAs and proteins. In the ribosome, the adequate assembly of rRNAs and ribosomal protein subunits in the nucleolus ensures suitable translational activity, with protein synthesis tuned according to intracellular demands of energy production, replication, proliferation, and growth. Disruption in the regulatory control of PtscM and PtrnM can impair ribosome biogenesis and ribosome function. Ribosomal impairment may, in turn, impact the synthesis of proteins engaged in functions as varied as telomere maintenance, apoptosis, and DNA repair, as well as intersect with mitochondria and telomerase activity. These cellular processes often malfunction in carcinogenesis and senescence. Here we discuss regulatory mechanisms of PtscMs and PtrnMs on ribosomal function. We also address chemical modification in rRNAs and their impacts on cellular metabolism, replication control, and senescence. Further, we highlight similarities and differences of PtscMs and PtrnMs in ribosomal intermediates during aging and carcinogenesis. Understanding these regulatory mechanisms may uncover critical steps for the development of more efficient oncologic and anti-aging therapies.

Cellular Imprinting Proteomics Assay: A Novel Method for Detection of Neural and Ocular Disorders Applied to Congenital Zika Virus Syndrome.

Congenital Zika syndrome was first described due to increased incidence of congenital abnormalities associated with Zika virus (ZIKV) infection. Since the eye develops as part of the embryo central nervous system (CNS) structure, it becomes a specialized compartment able to display symptoms of neurodegenerative diseases and has been proposed as a noninvasive approach to the early diagnosis of neurological diseases. Ocular lesions result from defects that occurred during embryogenesis and can become apparent in newborns exposed to ZIKV. Furthermore, the absence of microcephaly cannot exclude the occurrence of ocular lesions and other CNS manifestations. Considering the need for surveillance of newborns and infants with possible congenital exposure, we developed a method termed cellular imprinting proteomic assay (CImPA) to evaluate the ocular surface proteome specific to infants exposed to ZIKV during gestation compared to nonexposure. CImPA combines surface cells and fluid capture using membrane disks and a large-scale quantitative proteomics approach, which allowed the first-time report of molecular alterations such as neutrophil degranulation, cell death signaling, ocular and neurological pathways, which are associated with ZIKV infection with and without the development of congenital Zika syndrome, CZS. Particularly, infants exposed to ZIKV during gestation and without early clinical symptoms could be detected using the CImPA method. Lastly, this methodology has broad applicability as it could be translated in the study of several neurological diseases to identify novel diagnostic biomarkers. Data are available via ProteomeXchange with identifier PXD014038.

Characterization of Cerebellum-Specific Ribosomal DNA Epigenetic Modifications in Alzheimer's Disease: Should the Cerebellum Serve as a Control Tissue After All?

Alzheimer's disease (AD) is a neurodegenerative disease, known as the most common form of dementia. In AD onset, abnormal rRNA expression has been reported to be linked in pathogenesis. Although region-specific expression patterns have previously been reported in AD, it is not until recently that the cerebellum has come under the spotlight. Specifically, it is unclear whether DNA methylation is the mechanism involved in rRNA expression regulation in AD. Hence, we sought to explore the rDNA methylation pattern of two different brain regions - auditory cortex and cerebellum - from AD and age-/sex-matched controls. Our results showed differential hypermethylation at an upstream CpG region to the rDNA promoter when comparing cerebellum controls to auditory cortex controls. This suggests a possible regulatory region from rDNA expression regulation. Moreover, when comparing between AD and control cerebellum samples, we observed hypermethylation of the rDNA promoter region as well as an increase in rDNA content. In addition, we also observed increased rRNA levels in AD compared to control cerebellum. Although still considered a pathology-free brain region, there are growing findings that continue to suggest otherwise. Indeed, cerebellum from AD has been recently described as affected by the disease, presenting a unique pattern of molecular alterations. Given that we observed that increased rDNA promoter methylation did not silence rDNA gene expression, we suggest that rDNA promoter hypermethylation is playing a protective role in rDNA genomic stability and, therefore, increasing rRNA levels in AD cerebellum.

Exposure to an Environmentally Relevant Phthalate Mixture During Prostate Development Induces MicroRNA Upregulation and Transcriptome Modulation in Rats.

Environmental exposure to phthalates during intrauterine development might increase susceptibility to neoplasms in reproductive organs such as the prostate. Although studies have suggested an increase in prostatic lesions in adult animals submitted to perinatal exposure to phthalates, the molecular pathways underlying these alterations remain unclear. Genome-wide levels of mRNAs and miRNAs were monitored with RNA-seq to determine if perinatal exposure to a phthalate mixture in pregnant rats is capable of modifying gene expression during prostate development of the filial generation. The mixture contains diethyl-phthalate, di-(2-ethylhexyl)-phthalate, dibutyl-phthalate, di-isononyl-phthalate, di-isobutyl-phthalate, and benzylbutyl-phthalate. Pregnant females were divided into 4 groups and orally dosed daily from GD10 to PND21 with corn oil (Control: C) or the phthalate mixture at 3 doses (20 µg/kg/day: T1; 200 µg/kg/day: T2; 200 mg/kg/day: T3). The phthalate mixture decreased anogenital distance, prostate weight, and decreased testosterone level at the lowest exposure dose at PND22. The mixture also increased inflammatory foci and focal hyperplasia incidence at PND120. miR-184 was upregulated in all treated groups in relation to control and miR-141-3p was only upregulated at the lowest dose. In addition, 120 genes were deregulated at the lowest dose with several of these genes related to developmental, differentiation, and oncogenesis. The data indicate that phthalate exposure at lower doses can cause greater gene expression modulation as well as other downstream phenotypes than exposure at higher doses. A significant fraction of the downregulated genes were predicted to be targets of miR-141-3p and miR-184, both of which were induced at the lower exposure doses.

Point-of-care ultrasound evaluation and puncture simulation of the internal jugular vein by medical students.

OBJECTIVES: To show that medical students can evaluate the internal jugular vein (IJV) and its anatomical variations after rapid and focused training. We also aimed to evaluate the success rate of IJV puncture in simulation following traditional techniques (TTs) and monitored via ultrasound (US). MATERIALS AND METHODS: Six medical students without experience with US were given 4 h of theoretical-practical training in US, and then evaluated the IJV and common carotid artery (CCA) of 105 patients. They also simulated a puncture of the IJV at a demarcated point, where a TT was theoretically performed. RESULTS: Adequate images were obtained from 95% of the patients; the IJV, on the right side, was more commonly found in the anterolateral position in relation to the CCA (38%). On the left side, the most commonly position observed was the anterior (36%). The caliber of the IJV relative to the CCA greatly varied. The success rate in the IJV puncture simulation, observed with US, by the TTs was 55%. CONCLUSION: The training of medical students to recognize large neck vessels is a simple, quick, and feasible task and that can be integrated into the undergraduate medical curriculum.

Copy number variation in the susceptibility to systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease with a strong genetic component and etiology characterized by chronic inflammation and autoantibody production. The purpose of this study was to ascertain copy number variation (CNV) in SLE using a case-control design in an admixed Brazilian population. The whole-genome detection of CNV was performed using Cytoscan HD array in SLE patients and healthy controls. The best CNV candidates were then evaluated by quantitative real-time PCR in a larger cohort or validated using droplet digital PCR. Logistic regression models adjusted for sex and ancestry covariates was applied to evaluate the association between CNV with SLE susceptibility. The data showed a synergistic effect between the FCGR3B and ADAM3A loci with the presence of deletions in both loci significantly increasing the risk to SLE (5.9-fold) compared to the deletion in the single FCGR3B locus (3.6-fold). In addition, duplications in these genes were indeed more frequent in healthy subjects, suggesting that high FCGR3B/ADAM3A gene copy numbers are protective factors against to disease development. Overall, 21 rare CNVs were identified in SLE patients using a four-step pipeline created for identification of rare variants. Furthermore, heterozygous deletions overlapping the CFHR4, CFHR5 and HLA-DPB2 genes were described for the first time in SLE patients. Here we present the first genome-wide CNV study of SLE patients in a tri-hybrid population. The results show that novel susceptibility loci to SLE can be found once the distribution of structural variants is analyzed throughout the whole genome.

Uncovering the evolutionary history of neo-XY sex chromosomes in the grasshopper Ronderosia bergii (Orthoptera, Melanoplinae) through satellite DNA analysis.

BACKGROUND: Neo-sex chromosome systems arose independently multiple times in evolution, presenting the remarkable characteristic of repetitive DNAs accumulation. Among grasshoppers, occurrence of neo-XY was repeatedly noticed in Melanoplinae. Here we analyzed the most abundant tandem repeats of R. bergii (2n = 22, neo-XY♂) using deep Illumina sequencing and graph-based clustering in order to address the neo-sex chromosomes evolution. RESULTS: The analyses revealed ten families of satDNAs comprising about ~1% of the male genome, which occupied mainly C-positive regions of autosomes. Regarding the sex chromosomes, satDNAs were recorded within centromeric or interstitial regions of the neo-X chromosome and four satDNAs occurred in the neo-Y, two of them being exclusive (Rber248 and Rber299). Using a combination of probes we uncovered five well-defined cytological variants for neo-Y, originated by multiple paracentric inversions and satDNA amplification, besides fragmented neo-Y. These neo-Y variants were distinct in frequency between embryos and adult males. CONCLUSIONS: The genomic data together with cytogenetic mapping enabled us to better understand the neo-sex chromosome dynamics in grasshoppers, reinforcing differentiation of neo-X and neo-Y and revealing the occurrence of multiple additional rearrangements involved in the neo-Y evolution of R. bergii. We discussed the possible causes that led to differences in frequency for the neo-Y variants between embryos and adults. Finally we hypothesize about the role of DNA satellites in R. bergii as well as putative historical events involved in the evolution of the R. bergii neo-XY.

Satellite DNAs are conserved and differentially transcribed among Gryllus cricket species.

Satellite DNA (satDNA) is an abundant class of non-coding repetitive DNA that is preferentially found as tandemly repeated arrays in gene-poor heterochromatin but is also present in gene-rich euchromatin. Here, we used DNA- and RNA-seq from Gryllus assimilis to address the content and transcriptional patterns of satDNAs. We also mapped RNA-seq libraries for other Gryllus species against the satDNAs found in G. assimilis and G. bimaculatus genomes to investigate their evolutionary conservation and transcriptional profiles in Gryllus. Through DNA-seq read clustering analysis using RepeatExplorer, dotplots analysis and fluorescence in situ hybridization mapping, we found that ∼4% of the G. assimilis genome is represented by 11 well-defined A + T-rich satDNA families. These are mainly located in heterochromatic areas, with some repeats able to form high-order repeat structures. By in silico transcriptional analysis we identified satDNAs that are conserved in Gryllus but differentially transcribed. The data regarding satDNA presence in G. assimilis genome were discussed in an evolutionary context, with transcriptional data enabling comparisons between sexes and across tissues when possible. We discuss hypotheses for the conservation and transcription of satDNAs in Gryllus, which might result from their role in sexual differentiation at the chromatin level, heterochromatin formation and centromeric function.

Biomarkers of genome instability and cancer epigenetics.

Tumorigenesis is a multistep process involving genetic and epigenetic alterations that drive somatic evolution from normal human cells to malignant derivatives. Collectively, genetic and epigenetic alterations might be combined into biomarkers for the assessment of risk, the detection of early stage tumors, and accurate tumor characterization before and after treatment. Recent efforts have provided systematic approaches to cancer genomics through the application of massive sequencing of specific tumor types. Here, we review biomarkers of genome instability and epigenetics. Cancer evolvability and adaptation emerge through genetic and epigenetic lesions of a variety of sizes and qualities-from point mutations and small insertions/deletions to large-scale chromosomal rearrangements, alterations in whole chromosome copy number, preferential allelic expression of cancer risk alleles, and mechanisms that increase tumor mutation rates. We also review specific epigenetic mechanisms that facilitate or hinder tumor adaptation, including DNA methylation, histone modification, nucleosome remodeling, transcription factor activity, and small non-coding RNAs. Given the complexity of the carcinogenic process, the challenge ahead will be to interpret disparate signals across hundreds of genes and summarize these signals into a single actionable diagnosis that translates into specific treatments. Another challenge is to refine preventive efforts through the identification of epigenetic processes that mediate increased cancer rates in individuals exposed to sources of toxic environmental stress and pollution, specially through development and early childhood.

More epigenetic hits than meets the eye: microRNAs and genes associated with the tumorigenesis of retinoblastoma.

Retinoblastoma (RB), a childhood neoplasia of the retinoblasts, can occur unilaterally or bilaterally, with one or multiple foci per eye. RB is associated with somatic loss of function of both alleles of the tumor suppressor gene RB1. Hereditary forms emerge due to germline loss of function mutations in RB1 alleles. RB has long been the prototypic "model" cancer ever since Knudson's "two-hit" hypothesis. However, a simple two-hit model for RB is challenged by an increasing number of studies documenting additional hits that contribute to RB development. Here we review the genetics and epigenetics of RB with a focus on the role of small non-coding RNAs (microRNAs) and on novel findings indicating the relevance of DNA methylation in the development and prognosis of this neoplasia. Studies point to an elaborated landscape of genetic and epigenetic complexity, in which a number of events and pahtways play crucial roles in the origin and prognosis of RB. These include roles for microRNAs, inprinted loci, and parent-of-origin contributions to RB1 regulation and RB progression. This complexity is also manifested in the structure of the RB1 locus itself: it includes numerous repetitive DNA segments and retrotransposon insertion elements, some of which are actively transcribed from the RB1 locus. Altogether, we conclude that RB1 loss of function represents the tip of an iceberg of events that determine RB development, progression, severity, and disease risk. Comprehensive assessment of personalized RB risk will require genetic and epigenetic evaluations beyond RB1 protein coding sequences.

Phylogenetic footprinting reveals extensive conservation of Sonic Hedgehog (SHH) regulatory elements.

Sonic Hedgehog (SHH) plays a fundamental role in numerous developmental processes including morphogenesis of limbs, nervous system, and teeth. Using a Bayesian alignment algorithm for phylogenetic footprinting we analyzed approximately 28 kb of noncoding DNA in the SHH locus of human and mouse. This showed that the length of conserved noncoding sequences (4196 nt) shared by these species was approximately 3 times larger than the SHH coding sequence (1386 nt). Most segments were located in introns (53%) or within 2-kb regions upstream (16%) or downstream (20%) of the first and last SHH codon. Even though regions more than 2 kb upstream or downstream of the first and last SHH codon represented 57% (16 kb) of the sequence compared, they accounted for only 11% (494 nt) of the total length of conserved noncoding segments. One region of 650 nt downstream of SHH was identified as a putative scaffold/matrix attachment region (SMAR). Human-mouse analysis was complemented by sequencing in apes, monkeys, rodents, and bats, thus further confirming the evolutionary conservation of some segments. Gel-shift assays indicated that conserved segments are targeted by nuclear proteins and showed differences between two cell types that expressed different levels of SHH, namely human endothelial cells and breast cancer cells. The relevance of these findings with respect to regulation of SHH expression during normal and pathologic development is discussed.

The lowest diploid number (2n = 16) yet found in any primate: Callicebus lugens (Humboldt, 1811).

Morphologic, molecular and karyologic analyses of Callicebus lugens (Humboldt, 1811) of known geographic origin supported the proposition that this is a valid species. Morphologic and morphometric analyses showed evident differences between C. lugens and two other related taxa of the same group (Callicebus purinus and Callicebus torquatus). Cytochrome b DNA analyses (maximum parsimony, neighbour joining and maximum likelihood) were congruent in showing a strong association between C. lugens and Callicebus sp. of the torquatus group in one branch and a sister branch further divided into two clades: one with species of the personatus group and another, with species of the moloch group. Karyotypic analysis showed that C. lugens has the lowest diploid chromosome number of the primate order (2n = 16). Comparisons with other congeneric species clearly supported the proposition that C. lugens is karyotypically similar to others of the torquatus group.