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Spontaneous cancers in companion dogs are robust models of human disease. Tracking tumor-specific immune responses in these models requires reagents to perform species-specific single cell T cell receptor sequencing (scTCRseq). scTCRseq and integration with scRNA data have not been demonstrated on companion dogs with cancer. Here, five healthy dogs, two dogs with T cell lymphoma and four dogs with melanoma are selected to demonstrate applicability of scTCRseq in a cancer immunotherapy setting. Single-cell suspensions of PBMCs or lymph node aspirates are profiled using scRNA and dog-specific scTCRseq primers. In total, 77,809 V(D)J-expressing cells are detected, with an average of 3498 (348 - 5,971) unique clonotypes identified per sample. In total, 29/34, 40/40, 22/22 and 9/9 known functional TRAV, TRAJ, TRBV and TRBJ gene segments are observed respectively. Pseudogene or otherwise defective gene segments are also detected supporting re-annotation of several as functional. Healthy dogs exhibit highly diverse repertoires, T cell lymphomas exhibit clonal repertoires, and vaccine-treated melanoma dogs are dominated by a small number of highly abundant clonotypes. scRNA libraries define large clusters of V(D)J-expressing CD8+ and CD4 + T cells. Dominant clonotypes observed in melanoma PBMCs are predominantly CD8 + T cells, with activated phenotypes, suggesting possible anti-tumor T cell populations.
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Receptores de Antígenos de Linfócitos T , Análise de Célula Única , Animais , Cães , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Melanoma/genética , Melanoma/imunologia , Melanoma/veterinária , Doenças do Cão/imunologia , Doenças do Cão/genética , Linfoma de Células T/imunologia , Linfoma de Células T/veterinária , Linfoma de Células T/genéticaRESUMO
The fetal brain of the mouse is thought to be dependent upon the placenta as a source of serotonin (5-hydroxytryptamine; 5-HT) and other factors. How factors reach the developing brain remains uncertain but are postulated here to be part of the cargo carried by placental extracellular vesicles (EV). We have analyzed the protein, catecholamine, and small RNA content of EV from mouse trophoblast stem cells (TSC) and TSC differentiated into parietal trophoblast giant cells (pTGC), potential primary purveyors of 5-HT. Current studies examined how exposure of mouse neural progenitor cells (NPC) to EV from either TSC or pTGC affect their transcriptome profiles. The EV from trophoblast cells contained relatively high amounts of 5-HT, as well as dopamine and norepinephrine, but there were no significant differences between EV derived from pTGC and from TSC. Content of miRNA and small nucleolar (sno)RNA, however, did differ according to EV source, and snoRNA were upregulated in EV from pTGC. The primary inferred targets of the microRNA (miRNA) from both pTGC and TSC were mRNA enriched in the fetal brain. NPC readily internalized EV, leading to changes in their transcriptome profiles. Transcripts regulated were mainly ones enriched in neural tissues. The transcripts in EV-treated NPC that demonstrated a likely complementarity with miRNA in EV were mainly up- rather than downregulated, with functions linked to neuronal processes. Our results are consistent with placenta-derived EV providing direct support for fetal brain development and being an integral part of the placenta-brain axis.
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Vesículas Extracelulares , MicroRNAs , Humanos , Gravidez , Feminino , Animais , Camundongos , Serotonina/metabolismo , Placenta/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Vesículas Extracelulares/metabolismo , Encéfalo/metabolismo , Trofoblastos/metabolismo , Células-Tronco/metabolismoRESUMO
Lake Hillier is a hypersaline lake known for its distinctive bright pink color. The cause of this phenomenon in other hypersaline sites has been attributed to halophiles, Dunaliella, and Salinibacter, however, a systematic analysis of the microbial communities, their functional features, and the prevalence of pigment-producing-metabolisms has not been previously studied. Through metagenomic sequencing and culture-based approaches, our results evidence that Lake Hillier is composed of a diverse set of microorganisms including archaea, bacteria, algae, and viruses. Our data indicate that the microbiome in Lake Hillier is composed of multiple pigment-producer microbes, including Dunaliella, Salinibacter, Halobacillus, Psychroflexus, Halorubrum, many of which are cataloged as polyextremophiles. Additionally, we estimated the diversity of metabolic pathways in the lake and determined that many of these are related to pigment production. We reconstructed complete or partial genomes for 21 discrete bacteria (N = 14) and archaea (N = 7), only 2 of which could be taxonomically annotated to previously observed species. Our findings provide the first metagenomic study to decipher the source of the pink color of Australia's Lake Hillier. The study of this pink hypersaline environment is evidence of a microbial consortium of pigment producers, a repertoire of polyextremophiles, a core microbiome and potentially novel species.
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Wastewater-based epidemiology (WBE) is an effective way of tracking the appearance and spread of SARS-COV-2 lineages through communities. Beginning in early 2021, we implemented a targeted approach to amplify and sequence the receptor binding domain (RBD) of SARS-COV-2 to characterize viral lineages present in sewersheds. Over the course of 2021, we reproducibly detected multiple SARS-COV-2 RBD lineages that have never been observed in patient samples in 9 sewersheds located in 3 states in the USA. These cryptic lineages contained between 4 to 24 amino acid substitutions in the RBD and were observed intermittently in the sewersheds in which they were found for as long as 14 months. Many of the amino acid substitutions in these lineages occurred at residues also mutated in the Omicron variant of concern (VOC), often with the same substitutions. One of the sewersheds contained a lineage that appeared to be derived from the Alpha VOC, but the majority of the lineages appeared to be derived from pre-VOC SARS-COV-2 lineages. Specifically, several of the cryptic lineages from New York City appeared to be derived from a common ancestor that most likely diverged in early 2020. While the source of these cryptic lineages has not been resolved, it seems increasingly likely that they were derived from long-term patient infections or animal reservoirs. Our findings demonstrate that SARS-COV-2 genetic diversity is greater than what is commonly observed through routine SARS-CoV-2 surveillance. Wastewater sampling may more fully capture SARS-CoV-2 genetic diversity than patient sampling and could reveal new VOCs before they emerge in the wider human population.
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COVID-19 , SARS-CoV-2 , Animais , Humanos , SARS-CoV-2/genética , Águas Residuárias , COVID-19/epidemiologia , Variação GenéticaRESUMO
Opioid drugs are commonly prescribed analgesic to pregnant women. Direct exposure to such drugs may slow gut motility, alter gut permeability, and affect the gut microbiome. While such drugs affect gut microbiome in infants, no study to date has determined whether developmental exposure to such drugs results in longstanding effects on gut microbiota and correspondingly on host responses. We hypothesized developmental exposure to oxycodone (OXY) leads to enduring effects on gut microbiota and such changes are associated with adult neurobehavioral and metabolic changes. Female mice were treated daily with 5 mg OXY/kg or saline solution (control [CTL]) for 2 weeks prior to breeding and then throughout gestation. Male and female offspring pups were weaned, tested with a battery of behavioral and metabolic tests, and fecal boli were collected adulthood (120 days of age). In females, relative abundance of Butyricimonas spp., Bacteroidetes, Anaeroplasma spp., TM7, Enterococcus spp., and Clostridia were greater in OXY versus CTL individuals. In males, relative abundance of Coriobacteriaceae, Roseburia spp., Sutterella spp., and Clostridia were elevated in OXY exposed individuals. Bacterial changes were also associated with predictive metabolite pathway alterations that also varied according to sex. In males and females, affected gut microbiota correlated with metabolic but not behavioral alterations. The findings suggest that developmental exposure to OXY leads to lasting effects on adult gut microbiota that might affect host metabolism, possibly through specific bacterial metabolites or other bacterial-derived products. Further work is needed to characterize how developmental exposure to OXY affects host responses through the gut microbiome. IMPORTANCE This is the first work to show in a rodent model that in utero exposure to an opioid drug can lead to longstanding effects on the gut microbiota when examined at adulthood. Further, such bacterial changes are associated with metabolic host responses. Given the similarities between rodent and human microbiomes, it raises cause for concern that similar effects may become evident in children born to mothers taking oxycodone and other opioid drugs.
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Microbioma Gastrointestinal , Microbiota , Humanos , Adulto , Criança , Masculino , Feminino , Animais , Camundongos , Gravidez , Oxicodona/efeitos adversos , Analgésicos Opioides/efeitos adversos , Comportamento Social , BactériasRESUMO
Aim: To determine small RNA expression changes in mouse placenta induced by bisphenol A (BPA) exposure. Methods: Exposing female mice to BPA two weeks prior to conception through gestational day 12.5; whereupon fetal placentas were collected, frozen in liquid nitrogen and stored at -80°C. Small RNAs were isolated and used for small RNA-sequencing. Results: 43 small RNAs were differentially expressed. Target mRNAs were closely aligned to those expressed by thymus and brain, and pathway enrichment analyses indicated that such target mRNAs regulate neurogenesis and associated neurodevelopment processes. Conclusions: BPA induces several small RNAs in mouse placenta that might provide biomarkers for BPA exposure. Further, the placenta might affect fetal brain development through the secretion of miRNAs.
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Disruptores Endócrinos , MicroRNAs , Animais , Compostos Benzidrílicos/toxicidade , Feminino , Humanos , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Fenóis/toxicidade , Placenta/metabolismo , GravidezRESUMO
INTRODUCTION: The mouse placenta accumulates and possibly produces serotonin (5-hydroxytryptamine; 5-HT) in parietal trophoblast giant cells (pTGC) located at the interface between the placenta and maternal deciduum. However, the roles of 5-HT in placental function are unclear. This lack of information is unfortunate, given that selective serotonin-reuptake inhibitors are commonly used to combat depression in pregnant women. The high affinity 5-HT transporter SLC6A4 (also known as SERT) is the target of such drugs and likely controls much of 5-HT uptake into pTGC and other placental cells. We hypothesized that ablation of the Slc6a4 gene would result in morphological changes correlated with placental gene expression changes, especially for those involved in nutrient acquisition and metabolism, and thereby, provide insights into 5-HT placental function. METHODS: Placentas were collected at embryonic age (E) 12.5 from Slc6a4 knockout (KO) and wild-type (WT) conceptuses. Histological analyses, RNAseq, qPCR, and integrative correlation analyses were performed. RESULTS: Slc6a4 KO placentas had a considerable increased pTGC to spongiotrophoblast area ratio relative to WT placentas and significantly elevated expression of genes associated with intestinal functions, including nutrient sensing, uptake, and catabolism, and blood clotting. Integrative correlation analyses revealed upregulation of many of these genes was correlated with pTGC layer expansion. One other key gene was dopa decarboxylase (Ddc), which catalyzes conversion of L-5-hydroxytryptophan to 5-HT. DISCUSSION: Our studies possibly suggest a new paradigm relating to how 5-HT operates in the placenta, namely as a factor regulating metabolic functions and blood coagulation. We further suggest that pTGC might be functional analogs of enterochromaffin 5-HT-positive cells of the intestinal mucosa, which regulate similar activities within the gut. Further work, including proteomics and metabolomic studies, are needed to buttress our hypothesis.
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Placenta/fisiologia , Proteínas da Membrana Plasmática de Transporte de Serotonina/deficiência , Serotonina/fisiologia , Animais , Dopa Descarboxilase/genética , Dopa Descarboxilase/metabolismo , Feminino , Células Gigantes/fisiologia , Intestinos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Placenta/química , Placenta/citologia , Gravidez , RNA/análise , Análise de Sequência de RNA , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética , Proteínas da Membrana Plasmática de Transporte de Serotonina/fisiologia , Trofoblastos/citologia , Regulação para CimaRESUMO
Histone proteins undergo various modifications that alter chromatin structure, including addition of methyl groups. Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase that methylates lysine residue 27, and thereby suppresses gene expression. EZH2 plays integral roles in the uterus and other reproductive organs. We have previously shown that conditional deletion of uterine EZH2 results in increased proliferation of luminal and glandular epithelial cells, and RNA-seq analyses reveal several uterine transcriptomic changes in Ezh2 conditional (c) knockout (KO) mice that can affect estrogen signaling pathways. To pinpoint the origin of such gene expression changes, we used the recently developed spatial transcriptomics (ST) method with the hypotheses that Ezh2cKO mice would predominantly demonstrate changes in epithelial cells and/or ablation of this gene would disrupt normal epithelial/stromal gene expression patterns. Uteri were collected from ovariectomized adult WT and Ezh2cKO mice and analyzed by ST. Asb4, Cxcl14, Dio2, and Igfbp5 were increased, Sult1d1, Mt3, and Lcn2 were reduced in Ezh2cKO uterine epithelium vs. WT epithelium. For Ezh2cKO uterine stroma, differentially expressed key hub genes included Cald1, Fbln1, Myh11, Acta2, and Tagln. Conditional loss of uterine Ezh2 also appears to shift the balance of gene expression profiles in epithelial vs. stromal tissue toward uterine epithelial cell and gland development and proliferation, consistent with uterine gland hyperplasia in these mice. Current findings provide further insight into how EZH2 may selectively affect uterine epithelial and stromal compartments. Additionally, these transcriptome data might provide mechanistic understanding and valuable biomarkers for human endometrial disorders with epigenetic underpinnings.
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Proteína Potenciadora do Homólogo 2 de Zeste/genética , Camundongos/genética , Transcriptoma , Útero/metabolismo , Animais , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Feminino , Perfilação da Expressão Gênica , Camundongos/metabolismo , Camundongos KnockoutRESUMO
One model to study the emergence of the human trophoblast (TB) has been the exposure of pluripotent stem cells to bone morphogenetic protein 4 (BMP4) in presence of inhibitors of ACTIVIN/TGFB; A83-01 and FGF2; PD173074 (BAP), which generates a mixture of cytotrophoblast, syncytiotrophoblast, and cells with similarities to extravillous trophoblast. Here, H1 human embryonic stem cells were BAP-exposed under two O2 conditions (20% and 5%, respectively). At day 8, single nuclei RNA sequencing was used for transcriptomics analysis, thereby allowing profiling of fragile syncytial structures as well as the more resilient mononucleated cells. Following cluster analysis, two major groupings, one comprised of five (2,4,6,7,8) and the second of three (1,3,5) clusters were evident, all of which displayed recognized TB markers. Of these, two (2 and 3) weakly resembled extravillous trophoblast, two (5 and 6) strongly carried the hallmark transcripts of syncytiotrophoblast, while the remaining five were likely different kinds of mononucleated cytotrophoblast. We suggest that the two populations of nuclei within syncytiotrophoblast may have arisen from fusion events involving two distinct species of precursor cells. The number of differentially expressed genes between O2 conditions varied among the clusters, and the number of genes upregulated in cells cultured under 5% O2 was highest in syncytiotrophoblast cluster 6. In summary, the BAP model reveals an unexpectedly complex picture of trophoblast lineage emergence that will need to be resolved further in time-course studies.
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Developmental exposure to endocrine disrupting chemicals (EDCs), e.g., bisphenol A (BPA) or genistein (GEN), causes longstanding epigenome effects. MicroRNAs (miRs) regulate which mRNAs will be translated to proteins and thereby serve as the final checkpoint in epigenetic control. Scant amount is known, however, whether EDCs affect neural miRNA (miR) patterns. We aimed to test the hypothesis that developmental exposure of California mice (Peromyscus californicus) to GEN, BPA, or both chemicals influences hypothalamic miR/small RNA profiles and ascertain the extent such biomolecular alterations correlate with behavioral and metabolic changes. California mice were developmentally exposed to GEN (250 mg/kg feed weight, FW), GEN (250 mg/kg FW)+BPA (5 mg/kg FW), low dose (LD) BPA (5 mg/kg FW), or upper dose (UD) BPA (50 mg/kg FW). Adult offspring were tested in a battery of behavioral and metabolic tests; whereupon, mice were euthanized, brains were collected and frozen, small RNAs were isolated from hypothalamic punches, and subsequently sequenced. California mice exposed to one or both EDCs engaged in one or more repetitive behaviors. GEN, LD BPA, and UD BPA altered aspects of ultrasonic and audible vocalizations. Each EDC exposure led to sex-dependent differences in differentially expressed miR/small RNAs with miR7-2, miR146, and miR148a being increased in all female and male EDC exposed groups. Current findings reveal that developmental exposure to GEN and/or BPA affects hypothalamic miR/small RNA expression patterns, and such changes correlate with EDC-induced behavioral and metabolic alterations. miR146 is likely an important mediator and biomarker of EDC exposure in mammals, including humans.
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Disruptores Endócrinos , MicroRNAs , Animais , Compostos Benzidrílicos/toxicidade , Disruptores Endócrinos/toxicidade , Feminino , Hipotálamo , Masculino , Camundongos , MicroRNAs/genética , Peromyscus , Caracteres SexuaisRESUMO
INTRODUCTION: Pregnant women are increasingly being prescribed and abusing opioid drugs. As the primary communication organ between mother and conceptus, the placenta may be vulnerable to opioid effects but also holds the key to better understanding how these drugs affect long-term offspring health. We hypothesized that maternal treatment with oxycodone (OXY), the primary opioid at the center of the current crisis, deleteriously affects placental structure and gene expression patterns. METHODS: Female mice were treated daily with 5 mg OXY/kg or saline solution (Control, CTL) for two weeks prior to breeding and until placenta were collected at embryonic age 12.5. A portion of the placenta was fixed for histology, and the remainder was frozen for RNA isolation followed by RNAseq. RESULTS: Maternal OXY treatment reduced parietal trophoblast giant cell (pTGC) area and decreased the maternal blood vessel area within the labyrinth region. OXY exposure affected placental gene expression profiles in a sex dependent manner with female placenta showing up-regulation of many placental enriched genes, including Ceacam11, Ceacam14, Ceacam12, Ceacam13, Prl7b1, Prl2b1, Ctsq, and Tpbpa. In contrast, placenta of OXY exposed males had alteration of many ribosomal proteins. Weighted correlation network analysis revealed that in OXY female vs. CTL female comparison, select modules correlated with OXY-induced placental histological changes. Such associations were lacking in the male OXY vs. CTL male comparison. DISCUSSION: Results suggest OXY exposure alters placental histology. In response to OXY exposure, female placenta responds by upregulating placental enriched transcripts that are either unchanged or downregulated in male placenta. Such changes may shield female offspring from developmental origins of health and disease-based diseases.
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Analgésicos Opioides/efeitos adversos , Oxicodona/efeitos adversos , Placenta/efeitos dos fármacos , Animais , Feminino , Masculino , Camundongos , Placenta/metabolismo , Gravidez , Taxa de Gravidez , Razão de Masculinidade , Transcriptoma/efeitos dos fármacosRESUMO
The objective of this study was to evaluate the effects of increased PCR cycle number on sequencing results from samples with low microbial biomass, including bovine milk, and murine pelage and blood. We hypothesized that subjecting DNA from such samples to higher PCR cycle numbers would increase 16S rRNA sequencing coverage. DNA was extracted from matched samples of each type and multiple PCR cycle numbers were evaluated to generate a total of 96 libraries from 24 milk samples, 46 libraries from 23 pelage samples, and 170 libraries from 85 blood samples. 16S rRNA sequencing was performed on the Illumina MiSeq platform, and the coverage per sample, detected richness, and beta-diversity were evaluated. Across all sample types, higher PCR cycle numbers were associated with increased coverage. Surprisingly however, while higher PCR cycle numbers resulted in greater number of useable datapoints, no differences were detected in metrics of richness or beta-diversity. While reagent controls amplified for 40 cycles yielded similarly increased coverage, control and experimental samples were clearly differentiated based on beta-diversity. The results from this study support the use of higher PCR cycle numbers to evaluate samples with low microbial biomass.
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Bactérias , Sangue/microbiologia , Leite/microbiologia , Reação em Cadeia da Polimerase/métodos , RNA Bacteriano/isolamento & purificação , RNA Ribossômico 16S/isolamento & purificação , Animais , Bactérias/genética , Bactérias/isolamento & purificação , Bovinos , CamundongosRESUMO
Xenoestrogens are chemicals found in plant products, such as genistein (GEN), and in industrial chemicals, e.g., bisphenol A (BPA), present in plastics and other products that are prevalent in the environment. Early exposure to such endocrine disrupting chemicals (EDC) may affect brain development by directly disrupting neural programming and/or through the microbiome-gut-brain axis. To test this hypothesis, California mice (Peromyscus californicus) offspring were exposed through the maternal diet to GEN (250 mg/kg feed weight) or BPA (5 mg/kg feed weight, low dose- LD or 50 mg/kg, upper dose-UD), and dams were placed on these diets two weeks prior to breeding, throughout gestation, and lactation. Various behaviors, gut microbiota, and fecal metabolome were assessed at 90 days of age. The LD but not UD of BPA exposure resulted in individuals spending more time engaging in repetitive behaviors. GEN exposed individuals were more likely to exhibit such behaviors and showed socio-communicative disturbances. BPA and GEN exposed females had increased number of metabolites involved in carbohydrate metabolism and synthesis. Males exposed to BPA or GEN showed alterations in lysine degradation and phenylalanine and tyrosine metabolism. Current findings indicate cause for concern that developmental exposure to BPA or GEN might affect the microbiome-gut-brain axis.
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Compostos Benzidrílicos/toxicidade , Encéfalo/efeitos dos fármacos , Disbiose/induzido quimicamente , Disruptores Endócrinos/toxicidade , Microbioma Gastrointestinal/efeitos dos fármacos , Genisteína/toxicidade , Peromyscus/microbiologia , Fenóis/toxicidade , Efeitos Tardios da Exposição Pré-Natal , Animais , Transtorno do Espectro Autista/induzido quimicamente , Bactérias/efeitos dos fármacos , Bactérias/isolamento & purificação , Encéfalo/embriologia , Encéfalo/crescimento & desenvolvimento , Dieta , Modelos Animais de Doenças , Fezes/microbiologia , Feminino , Lactação , Masculino , Aprendizagem em Labirinto , Transtornos da Memória/induzido quimicamente , Metaboloma/efeitos dos fármacos , Peromyscus/embriologia , Peromyscus/crescimento & desenvolvimento , Peromyscus/metabolismo , Lesões Pré-Concepcionais/induzido quimicamente , Gravidez , Complicações na Gravidez/induzido quimicamente , Complicações na Gravidez/microbiologia , Comportamento Social , Especificidade da Espécie , Vocalização AnimalRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Placental trophoblast cells are potentially at risk from circulating endocrine-disrupting chemicals, such as bisphenol A (BPA). To understand how BPA and the reputedly more inert bisphenol S (BPS) affect the placenta, C57BL6J mouse dams were fed 200 µg/kg body weight BPA or BPS daily for 2 wk and then bred. They continued to receive these chemicals until embryonic day 12.5, whereupon placental samples were collected and compared with unexposed controls. BPA and BPS altered the expression of an identical set of 13 genes. Both exposures led to a decrease in the area occupied by spongiotrophoblast relative to trophoblast giant cells (GCs) within the junctional zone, markedly reduced placental serotonin (5-HT) concentrations, and lowered 5-HT GC immunoreactivity. Concentrations of dopamine and 5-hydroxyindoleacetic acid, the main metabolite of serotonin, were increased. GC dopamine immunoreactivity was increased in BPA- and BPS-exposed placentas. A strong positive correlation between 5-HT+ GCs and reductions in spongiotrophoblast to GC area suggests that this neurotransmitter is essential for maintaining cells within the junctional zone. In contrast, a negative correlation existed between dopamine+ GCs and reductions in spongiotrophoblast to GC area ratio. These outcomes lead to the following conclusions. First, BPS exposure causes almost identical placental effects as BPA. Second, a major target of BPA/BPS is either spongiotrophoblast or GCs within the junctional zone. Third, imbalances in neurotransmitter-positive GCs and an observed decrease in docosahexaenoic acid and estradiol, also occurring in response to BPA/BPS exposure, likely affect the placental-brain axis of the developing mouse fetus.
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Compostos Benzidrílicos/toxicidade , Encéfalo/efeitos dos fármacos , Disruptores Endócrinos/toxicidade , Fenóis/toxicidade , Sulfonas/toxicidade , Trofoblastos/efeitos dos fármacos , Animais , Dopamina/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Serotonina/metabolismo , Trofoblastos/metabolismoRESUMO
Human offspring encounter high amounts of phytoestrogens, such as genistein (GEN), through maternal diet and soy-based formulas. Such chemicals can exert estrogenic activity and thereby disrupt neurobehavioral programming. Besides inducing direct host effects, GEN might cause gut dysbiosis and alter gut metabolites. To determine whether exposure to GEN affects these parameters, California mice (Peromyscus californicus) dams were placed 2 weeks prior to breeding and throughout gestation and lactation on a diet supplemented with GEN (250 mg/kg feed weight) or AIN93G phytoestrogen-free control diet (AIN). At weaning, offspring socio-communicative behaviors, gut microbiota and metabolite profiles were assayed. Exposure of offspring to GEN-induced sex-dependent changes in gut microbiota and metabolites. GEN exposed females were less likely to investigate a novel female mouse when tested in a three-chamber social test. When isolated, GEN males and females exhibited increased latency to elicit their first call, suggestive of reduced motivation to communicate with other individuals. Correlation analyses revealed interactions between GEN-induced microbiome, metabolome and socio-communicative behaviors. Comparison of GEN males with AIN males revealed the fraction of calls above 20 kHz was associated with daidzein, α-tocopherol, Flexispira spp. and Odoribacter spp. Results suggest early GEN exposure disrupts normal socio-communicative behaviors in California mice, which are otherwise evident in these social rodents. Such effects may be due to GEN disruptions on neural programming but might also be attributed to GEN-induced microbiota shifts and resultant changes in gut metabolites. Findings indicate cause for concern that perinatal exposure to GEN may detrimentally affect the offspring microbiome-gut-brain axis.
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Encéfalo/efeitos dos fármacos , Microbioma Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/efeitos dos fármacos , Genisteína/farmacologia , Efeitos Tardios da Exposição Pré-Natal/fisiopatologia , Comunicação Animal , Animais , Encéfalo/fisiologia , Feminino , Microbioma Gastrointestinal/fisiologia , Trato Gastrointestinal/metabolismo , Trato Gastrointestinal/microbiologia , Humanos , Masculino , Peromyscus , Fitoestrógenos/farmacologia , Gravidez , Comportamento SocialRESUMO
BACKGROUND: Brain sexual differentiation is sculpted by precise coordination of steroid hormones during development. Programming of several brain regions in males depends upon aromatase conversion of testosterone to estrogen. However, it is not clear the direct contribution that Y chromosome associated genes, especially sex-determining region Y (Sry), might exert on brain sexual differentiation in therian mammals. Two species of spiny rats: Amami spiny rat (Tokudaia osimensis) and Tokunoshima spiny rat (T. tokunoshimensis) lack a Y chromosome/Sry, and these individuals possess an XO chromosome system in both sexes. Both Tokudaia species are highly endangered. To assess the neural transcriptome profile in male and female Amami spiny rats, RNA was isolated from brain samples of adult male and female spiny rats that had died accidentally and used for RNAseq analyses. RESULTS: RNAseq analyses confirmed that several genes and individual transcripts were differentially expressed between males and females. In males, seminal vesicle secretory protein 5 (Svs5) and cytochrome P450 1B1 (Cyp1b1) genes were significantly elevated compared to females, whereas serine (or cysteine) peptidase inhibitor, clade A, member 3 N (Serpina3n) was upregulated in females. Many individual transcripts elevated in males included those encoding for zinc finger proteins, e.g. zinc finger protein X-linked (Zfx). CONCLUSIONS: This method successfully identified several genes and transcripts that showed expression differences in the brain of adult male and female Amami spiny rat. The functional significance of these findings, especially differential expression of transcripts encoding zinc finger proteins, in this unusual rodent species remains to be determined.
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Encéfalo/metabolismo , Murinae/genética , Caracteres Sexuais , Transcriptoma , Animais , Feminino , Perfilação da Expressão Gênica , Masculino , Murinae/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de RNA , Cromossomo YRESUMO
Due to their antimicrobial properties, silver nanoparticles (AgNPs) are being used in non-edible and edible consumer products. It is not clear though if exposure to these chemicals can exert toxic effects on the host and gut microbiome. Conflicting studies have been reported on whether AgNPs result in gut dysbiosis and other changes within the host. We sought to examine whether exposure of Sprague-Dawley male rats for two weeks to different shapes of AgNPs, cube (AgNC) and sphere (AgNS) affects gut microbiota, select behaviors, and induces histopathological changes in the gastrointestinal system and brain. In the elevated plus maze (EPM), AgNS-exposed rats showed greater number of entries into closed arms and center compared to controls and those exposed to AgNC. AgNS and AgNC treated groups had select reductions in gut microbiota relative to controls. Clostridium spp., Bacteroides uniformis, Christensenellaceae, and Coprococcus eutactus were decreased in AgNC exposed group, whereas, Oscillospira spp., Dehalobacterium spp., Peptococcaeceae, Corynebacterium spp., Aggregatibacter pneumotropica were reduced in AgNS exposed group. Bacterial reductions correlated with select behavioral changes measured in the EPM. No significant histopathological changes were evident in the gastrointestinal system or brain. Findings suggest short-term exposure to AgNS or AgNC can lead to behavioral and gut microbiome changes.
Assuntos
Disbiose/microbiologia , Microbioma Gastrointestinal/efeitos dos fármacos , Nanopartículas Metálicas/efeitos adversos , Aggregatibacter/efeitos dos fármacos , Animais , Bacteroides/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/fisiopatologia , Clostridium/efeitos dos fármacos , Corynebacterium/efeitos dos fármacos , Disbiose/induzido quimicamente , Disbiose/fisiopatologia , Fezes/microbiologia , Microbioma Gastrointestinal/genética , Trato Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/fisiopatologia , Humanos , Nanopartículas Metálicas/administração & dosagem , Peptococcus/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
Bisphenol A (BPA) is an endocrine-disrupting chemical (EDC) prevalent in many household items. Rodent models and human epidemiological studies have linked this chemical to neurobehavior impairments. In California mice, developmental exposure to BPA results in sociosexual disorders at adulthood, including communication and biparental care deficits, behaviors that are primarily regulated by the hypothalamus. Thus, we sought to examine the transcriptomic profile in this brain region of juvenile male and female California mice offspring exposed from periconception through lactation to BPA or ethinyl estradiol (EE, estrogen present in birth control pills and considered a positive estrogen control for BPA studies). Two weeks prior to breeding, P0 females were fed a control diet, or this diet supplemented with 50 mg BPA/kg feed weight or 0.1 ppb EE, and continued on the diets through lactation. At weaning, brains from male and female offspring were collected, hypothalamic RNA isolated, and RNA-seq analysis performed. Results indicate that BPA and EE groups clustered separately from controls with BPA and EE exposure leading to unique set of signature gene profiles. Kcnd3 was downregulated in the hypothalamus of BPA- and EE-exposed females, whereas Tbl2, Topors, Kif3a, and Phactr2 were upregulated in these groups. Comparison of transcripts differentially expressed in BPA and EE groups revealed significant enrichment of gene ontology terms associated with microtubule-based processes. Current results show that perinatal exposure to BPA or EE can result in several transcriptomic alterations, including those associated with microtubule functions, in the hypothalamus of California mice. It remains to be determined whether these genes mediate BPA-induced behavioral disruptions.
Assuntos
Compostos Benzidrílicos/toxicidade , Disruptores Endócrinos/toxicidade , Etinilestradiol/toxicidade , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Fenóis/toxicidade , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Transcriptoma/efeitos dos fármacos , Animais , Regulação para Baixo , Feminino , Masculino , Exposição Materna , Peromyscus , Gravidez , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Regulação para CimaRESUMO
Developmental exposure of turtles and other reptiles to endocrine-disrupting chemicals (EDCs), including bisphenol A (BPA) and ethinyl estradiol (EE), can stimulate partial to full gonadal sex-reversal in males. We have also recently shown that in ovo exposure to either EDC can induce similar sex-dependent behavioral changes typified by improved spatial learning and memory or possibly feminized brain responses. Observed behavioral changes are presumed to be due to BPA- and EE-induced brain transcriptomic alterations during development. To test this hypothesis, we treated painted turtles (Chrysemys picta) at developmental stage 17, incubated at 26°C (male-inducing temperature), with 1) BPA (1 ng/µl), 2) EE (4 ng/µl), or 3) vehicle ethanol (control group). Ten months after hatching and completion of the behavioral tests, juvenile turtles were euthanized, brains were collected and frozen in liquid nitrogen, and RNA was isolated for RNA-Seq analysis. Turtles exposed to BPA clustered separately from EE-exposed and control individuals. More transcripts and gene pathways were altered in BPA vs. EE individuals. The one transcript upregulated in both BPA- and EE-exposed individuals was the mitochondrial-associated gene, ND5, which is involved in oxidative phosphorylation. Early exposure of turtles to BPA increases transcripts linked with ribosomal and mitochondrial functions, especially bioenergetics, which has been previously linked with improved cognitive performance. In summary, even though both BPA and EE resulted in similar behavioral alterations, they diverge in the pattern of neural transcript alterations with early BPA significantly upregulating several genes involved in oxidative phosphorylation, mitochondrial activity, and ribosomal function, which could enhance cognitive performance.