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The utilization of transcriptomic studies identifying profiles of gene expression, especially in toxicogenomics, has catapulted next-generation sequencing to the forefront of reproductive toxicology. An innovative yet underutilized RNA sequencing technique emerging into this field is single-cell RNA sequencing (scRNA-seq), which provides sequencing at the individual cellular level of gonad tissue. ScRNA-seq provides a novel and unique perspective for identifying distinct cellular profiles, including identification of rare cell subtypes. The specificity of scRNA-seq is a powerful tool for reproductive toxicity research, especially for translational animal models including zebrafish. Studies to date not only have focused on 'tissue atlassing' or characterizing what cell types make up different tissues but have also begun to include toxicant exposure as a factor that this review aims to explore. Future scRNA-seq studies will contribute to understanding exposure-induced outcomes; however, the trade-offs with traditional methods need to be considered.
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BACKGROUND: Study results of prediagnostic dietary fat intake and breast cancer mortality have been inconclusive. While dietary fat subtypes [saturated (SFA), polyunsaturated (PUFA), and monounsaturated (MUFA) fatty acids] may have different biological effects, there is little evidence regarding the association of dietary fat and fat subtype intake with mortality after breast cancer diagnosis. METHODS: Women with incident, pathologically confirmed invasive breast cancer and complete dietary data (n = 793) were followed in a population-based study, the Western New York Exposures and Breast Cancer study. Usual intake before diagnosis of total fat and subtypes were estimated from a food frequency questionnaire completed at baseline. HRs and 95% confidence intervals (CI) for all-cause and breast cancer-specific mortality were estimated with Cox proportional hazards models. Interactions by menopausal status, estrogen receptor (ER) status, and tumor stage were examined. RESULTS: Median follow-up time was 18.75 years; 327 (41.2%) participants had died. Compared with lower intake, greater intake of total fat (HR, 1.05; 95% CI, 0.65-1.70), SFA (1.31; 0.82-2.10), MUFA (0.99; 0.61-1.60), and PUFA (0.99; 0.56-1.75) was not associated with breast cancer-specific mortality. There was also no association with all-cause mortality. Results did not vary by menopausal status, ER status, or tumor stage. CONCLUSIONS: Prediagnostic intake of dietary fat and fat subtypes was not associated with either all-cause or breast cancer mortality in a population-based cohort of breast cancer survivors. IMPACT: Understanding factors affecting survival among women diagnosed with breast cancer is critically important. Dietary fat intake prior to diagnosis may not impact that survival.
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Neoplasias da Mama , Gorduras Insaturadas na Dieta , Feminino , Humanos , Neoplasias da Mama/mortalidade , Dieta , Gorduras na Dieta , Ácidos Graxos , New York/epidemiologiaRESUMO
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent and environmentally persistent endocrine disrupting chemical. Our previous work demonstrated the latent reproductive maladies of early-life TCDD exposure in zebrafish. Zebrafish acutely exposed to low, environmentally relevant levels of TCDD (50 pg/mL) during two windows of sexual differentiation in development (1 hour of exposure at 3 and 7 weeks postfertilization) were later infertile, showed a reduction in sperm, and exhibited gene expression consistent with an altered microenvironment, even months after exposure. Due to the highly heterogeneous cell- type and -stage landscape of the testes, we hypothesized various cell types contribute markedly different profiles toward the pathology of TCDD exposure. To investigate the contributions of the diverse cell types in the adult zebrafish testes to TCDD-induced pathology, we utilized single-cell RNA-seq and the 10x Genomics platform. The method successfully captured every stage of testicular germ cell development. Testes of adult fish exposed during sexual differentiation to TCDD contained sharply decreased populations of late spermatocytes, spermatids, and spermatozoa. Spermatogonia and early spermatocyte populations were, in contrast, enriched following exposure. Pathway analysis of differentially expressed genes supported previous findings that TCDD exposure resulted in male infertility, and suggested this outcome is due to apoptosis of spermatids and spermatozoa, even years after exposure cessation. Increased germ cell apoptosis was confirmed histologically. These results provide support for an environmental exposure explanation of idiopathic male infertility.
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Per- and polyfluoroalkyl substances (PFASs) are ubiquitous in the environment and are tied to myriad health effects. Despite the phasing out of the manufacturing of two types of PFASs (perfluorosulfonic acid (PFOS) and perfluorooctanoic acid (PFOA)), chemical composition renders them effectively indestructible by ambient environmental processes, where they thus remain in water. Exposure via water can affect both human and aquatic wildlife. PFASs easily cross the placenta, exposing the fetus at critical windows of development. Little is known about the effects of low-level exposure during this period; even less is known about the potential for multi- and transgenerational effects. We examined the effects of ultra-low, very low, and low-level PFAS exposure (7, 70, and 700 ng/L PFOA; 24, 240, 2400 ng/L PFOS; and stepwise mixtures) from 0-5 days post-fertilization (dpf) on larval zebrafish (Danio rerio) mortality, morphology, behavior and gene expression and fecundity in adult F0 and F1 fish. As expected, environmentally relevant PFAS levels did not affect survival. Morphological abnormalities were not observed until the F1 and F2 generations. Behavior was affected differentially by each chemical and generation. Gene expression was increasingly perturbed in each generation but consistently showed lipid pathway disruption across all generations. Dysregulation of behavior and gene expression is heritable, even in larvae with no direct or indirect exposure. This is the first report of the transgenerational effects of PFOA, PFOS, and their mixture in terms of zebrafish behavior and untargeted gene expression.
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In this report, we compare the outcomes and limitations of two methods of transcriptomic inquiry on adult zebrafish testes exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during sexual differentiation: conventional or bulk RNA-seq (bulk-seq) and single cell RNA sequencing (scRNA-seq) data. scRNA-seq has emerged as a valuable tool for uncovering cell type-specific transcriptome dynamics which exist in heterogeneous tissue. Our lab previously showed the toxicological value of the scRNA-seq pipeline to characterize the sequelae of TCDD exposure in testes, demonstrating that loss of spermatids and spermatozoa, but not other cell types, contributed to the pathology of infertility in adult male zebrafish exposed during sexual differentiation. To investigate the potential for technical artifacts in scRNA-seq such as cell dissociation effects and reduced transcriptome coverage, we compared bulk-sequenced and scRNA-seq-paired samples from control and TCDD-exposed samples to understand what is gained and lost in scRNA-seq vs bulk-seq, both transcriptomically and toxicologically. We hypothesized that the testes may be sensitive to tissue disruption as they contain multiple cell types under constant division and/or maturation, and that TCDD exposure may mediate the extent of sensitivity. Thus, we sought to understand the extent to which this dissociation impacts the toxicological value of data returned from scRNA-seq. We confirm that the required dissociation of individual cells from intact tissue has a significant impact on gene expression, affecting gene pathways with the potential to confound toxicogenomics studies on exposures if findings are not well-controlled and well-situated in context. Additionally, a common scRNA-seq method using cDNA amplified from the 3' end of mRNA under-detects low-expressing transcripts including transcription factors. We confirm this, and show TCDD-related genes may be overlooked by scRNA-seq, however, this under-detection effect is not mediated by TCDD exposure. Even so, scRNA-seq generally extracted toxicologically relevant information better than the bulk-seq method in the present study. This report aims to inform future experimental design for transcriptomic investigation in the growing field of toxicogenomics by demonstrating the differential information extracted from sequencing cells-despite being from the same tissue and exposure scheme-is influenced by the specific protocol used, with implications for the interpretation of exposure-induced risk.
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Triclosan, triclocarban and 4-nonylphenol are all chemicals of emerging concern found in a wide variety of consumer products that have exhibited a wide range of endocrine-disrupting effects and are present in increasing amounts in groundwater worldwide. Results of the present study indicate that exposure to these chemicals at critical developmental periods, whether long-term or short-term in duration, leads to significant mortality, morphologic, behavioral and transcriptomic effects in zebrafish (Danio rerio). These effects range from total mortality with either long- or short-term exposure at 100 and 1000 nM of triclosan, to abnormalities in uninflated swim bladder seen with long-term exposure to triclocarban and short-term exposure to 4-nonylphenol, and cardiac edema seen with short-term 4-nonylphenol exposure. Additionally, a significant number of genes involved in neurological and cardiovascular development were differentially expressed after the exposures, as well as lipid metabolism genes and metabolic pathways after exposure to each chemical. Such changes in behavior, gene expression, and pathway abnormalities caused by these three known endocrine disruptors have the potential to impact not only the local ecosystem, but human health as well.
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Metformin is found in the majority of lakes and streams in the United States, leading to widespread environmental exposure. Results of the present study indicate that extended duration metformin exposure at critical developmental periods leads to decreased survival rates in zebrafish (danio rerio), an NIH approved human model. Significant abnormalities are seen with extended duration metformin exposure from 4 h post fertilization up to 5 days post fertilization, although short term metformin exposure for 24 h at 4-5 days post fertilization did not lead to any significant abnormalities. Both extended and short term duration did however have an impact on locomotor activity of zebrafish, and several genes involved in neurological and cardiovascular development were differentially expressed after exposure to metformin. The changes seen in behavior, gene expression and morphological abnormalities caused by metformin exposure should be examined further in future studies in order to assess their potential human health implications as metformin prescriptions continue to increase worldwide.
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Desenvolvimento Embrionário/efeitos dos fármacos , Metformina/toxicidade , Teratogênicos/toxicidade , Transcriptoma/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Peixe-Zebra , Animais , Comportamento Animal/efeitos dos fármacos , Osso e Ossos/anormalidades , Edema Cardíaco , Embrião não Mamífero/anormalidades , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/fisiologia , Feminino , Locomoção/efeitos dos fármacos , Masculino , Fenótipo , Peixe-Zebra/anormalidades , Peixe-Zebra/genética , Peixe-Zebra/fisiologiaRESUMO
Anthropogenic surface and ground water contamination by chemicals is a global problem, and there is an urgent need to develop tools to identify and elucidate biological effects. Contaminants of emerging concern (CECs) are not typically monitored or regulated and those with known or suspected endocrine disrupting potential have been termed endocrine disrupting chemicals (EDCs). Many CECs are known to be neurotoxic (e.g., insecticides) and many are incompletely characterized. Behavioral responses can identify chemicals with neuroactive properties, which can be relevant to EDC mechanisms (e.g., neuroendocrine disturbances). Two freshwater species, Daphnia pulex and Danio rerio, were evaluated for swimming behavior alterations resulting from 24-hr exposure to 9 CECs: triclosan, triclocarban, chlorpyrifos, dieldrin, 4-nonylphenol, bisphenol-A, atrazine, metformin, and estrone. This is the first step in the development of a bioassay for detecting estrogenic and/or anti-androgenic activity with the goal to evaluate complex mixtures of uncharacterized contaminants in water samples. The second step, described in a subsequent report, examines transcriptome alterations following chemical exposure. Significant differences in the swimming behavior response and sensitivity were found across chemicals within a species and across species for a given chemical in this unique optical bioassay system. In the concentration ranges studied, significant behavioral alterations were detected for 6 of 9 CECs for D. pulex and 4 of 9 CECs for D. rerio. These results underscore the utility of this bioassay to identify behavioral effects of sublethal concentrations of CECs before exploration of transcriptomic alterations for EDC detection.
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Disruptores Endócrinos , Poluentes Químicos da Água , Animais , Daphnia/genética , Disruptores Endócrinos/toxicidade , Estrona , Poluentes Químicos da Água/análise , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/genéticaRESUMO
Estrone and BPA are two endocrine disrupting chemicals (EDCs) that are predicted to be less potent than estrogens such as 17ß-estradiol and 17α-ethinylestradiol. Human exposure concentrations to estrone and BPA can be as low as nanomolar levels. However, very few toxicological studies have focused on the nanomolar-dose effects. Low level of EDCs can potentially cause non-monotonic responses. In addition, exposures at different developmental stages can lead to different health outcomes. To identify the nanomolar-dose effects of estrone and BPA, we used zebrafish modeling to study the phenotypic and transcriptomic responses after extended duration exposure from 0 to 5 days post-fertilization (dpf) and short-term exposure at days 4-5 post fertilization. We found that non-monotonic transcriptomic responses occurred after extended duration exposures at 1 nM of estrone or BPA. At this level, estrone also caused hypoactivity locomotive behavior in zebrafish. After both extended duration and short-term exposures, BPA led to more apparent phenotypic responses, i.e. skeletal abnormalities and locomotion changes, and more significant transcriptomic responses than estrone exposure. After short-term exposure, BPA at concentrations equal or above 100 nM affected locomotive behavior and changed the expression of both estrogenic and non-estrogenic genes that are linked to neurological diseases. These data provide gaps of mechanisms between neurological genes expression and associated phenotypic response due to estrone or BPA exposures. This study also provides insights for assessing the acceptable concentration of BPA and estrone in aquatic environments.
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Disruptores Endócrinos , Estrona , Animais , Compostos Benzidrílicos/toxicidade , Disruptores Endócrinos/toxicidade , Estrona/toxicidade , Humanos , Fenóis , Transcriptoma , Peixe-Zebra/genéticaRESUMO
Generation of reactive oxygen species, a critical factor in cisplatin-induced ototoxicity, leads to the formation of peroxynitrite, which in turn results in the nitration of susceptible proteins. Previous studies indicated that LMO4, a transcriptional regulator, is the most abundantly nitrated cochlear protein after cisplatin treatment and that LMO4 nitration facilitates ototoxicity in rodents. However, the role of this mechanism in regulating cisplatin-induced hair cell loss in non-mammalian models is unknown. As the mechanosensory hair cells in the neuromasts of zebrafish share many features with mammalian inner ear and is a good model for studying ototoxicity, we hypothesized that cisplatin treatment induces protein nitration and Lmo4 degradation in zebrafish hair cells, thereby facilitating hair cell loss. Immunostaining with anti-parvalbumin revealed a significant decrease in the number of hair cells in the neuromast of cisplatin treated larvae. In addition, cisplatin treatment induced a significant decrease in the expression of Lmo4 protein and a significant increase in nitrotyrosine levels, in the hair cells. The cisplatin-induced changes in Lmo4 and nitrotyrosine levels strongly correlated with hair cell loss, implying a potential link. Furthermore, a significant increase in the expression of activated Caspase-3 in zebrafish hair cells, post cisplatin treatment, suggested that cisplatin-induced decrease in Lmo4 levels is accompanied by apoptosis. These findings suggest that nitrative stress and Lmo4 degradation are important factors in cisplatin-induced hair cell loss in zebrafish neuromasts and that zebrafish could be used as a model to screen the otoprotective efficacy of compounds that inhibit protein nitration.
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Proteínas Adaptadoras de Transdução de Sinal , Antineoplásicos/toxicidade , Cisplatino/toxicidade , Células Ciliadas Auditivas/efeitos dos fármacos , Proteínas com Domínio LIM , Estresse Oxidativo/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Animais Geneticamente Modificados , Feminino , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas/patologia , Proteínas com Domínio LIM/metabolismo , Masculino , Estresse Oxidativo/fisiologia , Peixe-ZebraRESUMO
The legacy endocrine disrupting chemical and aryl hydrocarbon receptor agonist, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is produced as a byproduct of industrial processes and causes adverse health effects ranging from skin irritation to cancer. TCDD endpoints are also observed in subsequent, unexposed generations; however, the mechanisms of these multi- and transgenerational effects are unknown. We hypothesized an epigenetic mechanism, specifically DNA methylation for the transgenerational, male-mediated reproductive effects of developmental TCDD exposure. Using whole genome bisulfite sequencing, we evaluated DNA methylation changes in three generations of zebrafish, the first of which was exposed to TCDD during sexual development at 50 ppt for 1 h at both 3- and 7-week post-fertilization. We discovered that TCDD induces multi- and transgenerational methylomic changes in testicular tissue from zebrafish with decreased reproductive capacity, but most significantly in the indirectly exposed F1 generation. In comparing differentially methylated genes to concurrent transcriptomic changes, we identified several genes and pathways through which transgenerational effects of low level TCDD exposure are likely inherited. These include significant differential methylation of genes involved in reproduction, endocrine function, xenobiotic metabolism, and epigenetic processing. Notably, a number of histone modification genes were both differentially methylated and expressed in all generations, and many differentially methylated genes overlapped between multiple generations. Collectively, our results suggest that DNA methylation is a promising mechanism to explain male-mediated transgenerational reproductive effects of TCDD exposure in zebrafish, and these effects are likely inherited through integration of multiple epigenetic pathways.
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Microplastics (MPs) are a ubiquitous pollutant detected not only in marine and freshwater bodies, but also in tap and bottled water worldwide. While MPs have been extensively studied, the toxicity of their smaller counterpart, nanoplastics (NPs), is not well documented. Despite likely large-scale human and animal exposure to NPs, the associated health risks remain unclear, especially during early developmental stages. To address this, we investigated the health impacts of exposures to both 50 and 200 nm polystyrene NPs in larval zebrafish. From 6 to 120 h post-fertilization (hpf), developing zebrafish were exposed to a range of fluorescent NPs (10-10,000 parts per billion). Dose-dependent increases in accumulation were identified in exposed larval fish, potentially coinciding with an altered behavioral response as evidenced through swimming hyperactivity. Notably, exposures did not impact mortality, hatching rate, or deformities; however, transcriptomic analysis suggests neurodegeneration and motor dysfunction at both high and low concentrations. Furthermore, results of this study suggest that NPs can accumulate in the tissues of larval zebrafish, alter their transcriptome, and affect behavior and physiology, potentially decreasing organismal fitness in contaminated ecosystems. The uniquely broad scale of this study during a critical window of development provides crucial multidimensional characterization of NP impacts on human and animal health.
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Poluentes Químicos da Água , Peixe-Zebra/genética , Animais , Ecossistema , Embrião não Mamífero , Humanos , Larva , Microplásticos , Plásticos , TranscriptomaRESUMO
BACKGROUND: Clostridioides difficile infection and colonization are common in pediatric Crohn's disease (CD). Our aims were to test the relationship between C. difficile positivity and bowel resection surgery and to characterize microbial shifts associated with C. difficile carriage and surgery. METHODS: A retrospective single-center study of 75 pediatric CD patients tested for association between C. difficile carriage and bowel resection surgery. A prospective single-center study of 70 CD patients utilized C. difficile testing and shotgun metagenomic sequencing of fecal samples to define microbiota variation stratified by C. difficile carriage or history of surgery. RESULTS: The rate of bowel resection surgery increased from 21% in those without C. difficile to 67% in those with (P = 0.003). From a Kaplan-Meier survival model, the hazard ratio for time to first surgery was 4.4 (95% CI, 1.2-16.2; P = 0.00) in patients with positive C. difficile testing in the first year after diagnosis. Multivariable logistic regression analysis confirmed this association (odds ratio 16.2; 95% CI, 2.2-120; P = 0.006). Larger differences in microbial abundance and metabolic pathways were observed in patients with prior surgery than in those with C. difficile carriage. Depletion of Alistipes and Ruminococcus species and reduction in methionine biosynthesis were noted in patients with both C. difficile carriage and past surgery. CONCLUSIONS: A positive C. difficile test during the first year after diagnosis is associated with decreased time to first bowel resection surgery in pediatric Crohn's disease. Depletion of beneficial commensals and methionine biosynthesis in patients with C. difficile carriage may contribute to increased risk for surgery.
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Clostridioides difficile , Colectomia/estatística & dados numéricos , Doença de Crohn/microbiologia , Doença de Crohn/cirurgia , Enterocolite Pseudomembranosa/microbiologia , Enterocolite Pseudomembranosa/cirurgia , Adolescente , Criança , Fezes/microbiologia , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Metagenoma , Metionina/biossíntese , Modelos de Riscos Proporcionais , Estudos Prospectivos , Estudos Retrospectivos , Fatores de TempoRESUMO
Lead (Pb2+) is a major public health hazard for urban children, with profound and well-characterized developmental and behavioral implications across the lifespan. The ability of early Pb2+ exposure to induce epigenetic changes is well-established, suggesting that Pb2+-induced neurobehavioral deficits may be heritable across generations. Understanding the long-term and multigenerational repercussions of lead exposure is crucial for clarifying both the genotypic alterations behind these behavioral outcomes and the potential mechanism of heritability. To study this, zebrafish (Danio rerio) embryos (<2 h post fertilization; EK strain) were exposed for 24 h to waterborne Pb2+ at a concentration of 10 µM. This exposed F0 generation was raised to adulthood and spawned to produce the F1 generation, which was subsequently spawned to produce the F2 generation. Previous avoidance conditioning studies determined that a 10 µM Pb2+ dose resulted in learning impairments persisting through the F2 generation. RNA was extracted from control- and 10 µM Pb2+-lineage F2 brains, (n = 10 for each group), sequenced, and transcript expression was quantified utilizing Quant-Seq. 648 genes were differentially expressed in the brains of F2 lead-lineage fish versus F2 control-lineage fish. Pathway analysis revealed altered genes in processes including synaptic function and plasticity, neurogenesis, endocrine homeostasis, and epigenetic modification, all of which are implicated in lead-induced neurobehavioral deficits and/or their inheritance. These data will inform future investigations to elucidate the mechanism of adult-onset and transgenerational health effects of developmental lead exposure.
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Encéfalo/metabolismo , Chumbo/farmacologia , Transcriptoma/efeitos dos fármacos , Peixe-Zebra/genética , Animais , Sistema Endócrino/metabolismo , Epigênese Genética/efeitos dos fármacos , Feminino , Padrões de Herança/efeitos dos fármacos , Masculino , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismoRESUMO
Axolotls (Ambystoma mexicanum) from a research colony presented with multifocal, white chalky to gray skin lesions, a diffuse whitish to blue hue to the integument, and friable gill filaments. Skin scrapings and wet mounts revealed Chilodonella, Ichthyobodo, and a trichodinid species. The average overall burden (that is, all 3 species) per axolotl ranged from 0 to 25 parasites per 40 × field (p40f; mean ± 1 SD, 2.6 ± 5.5), with a prevalence of 12%, 60%, and 48%, respectively. Concurrent with husbandry modifications, axolotls were treated with an 8-h static immersion bath that contained 0.025 mL/L 37% formaldehyde. Chilodonella organisms were no longer observed after the initial treatment, and Ichthyobodo decreased from 2.4 ± 5.6 to 0.6 ± 1.8 organisms p40f. However, the average overall burden increased 4-fold to 10.5 ± 9.8 parasites p40f, and the trichodinid organisms increased 13-fold from 0.8 ± 2.3 to 10.4 ± 9.2 organisms p40f. A second treatment consisted of an 8-h immersion bath that contained 0.05 mL/L 37% formaldehyde on 2 consecutive days. A significant change was noted in the average overall burden of 0.5 ± 1.1 parasites p40f, a greater than 5- and 21-fold decrease from pretreatment and after the initial treatment, respectively. No significant change between the first and second treatment was observed for Ichthyobodo, with 0.6 ± 1.2 organisms p40f, but this number represented a significant decrease from pretreatment. After the second treatment, the trichodinid organism was detected in only one axolotl, with a low overall burden of 0.2 ± 0.4 organisms p40f and resulting in a significant decrease in the trichodinid count to 0.01 ± 0.04 organisms p40f. Treatment with formalin (37% formaldehyde), in conjunction with husbandry improvements, was effective in significantly reducing ectoparasite burden and eliminating clinical symptoms in axolotls but did not fully eliminate all protozoa.
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Ambystoma mexicanum/parasitologia , Ectoparasitoses/veterinária , Parasitos/classificação , Animais , Desinfetantes/uso terapêutico , Ectoparasitoses/tratamento farmacológico , Ectoparasitoses/parasitologia , Formaldeído/uso terapêutico , Ciência dos Animais de LaboratórioRESUMO
The small protein modifier ubiquitin regulates various aspects of cellular biology through its chemical conjugation onto proteins. Ubiquitination of proteins presents itself in numerous iterations, from a single mono-ubiquitination event to chains of poly-ubiquitin. Ubiquitin chains can be attached onto other proteins or can exist as unanchored species, i.e. free from another protein. Unanchored ubiquitin chains are thought to be deleterious to the cell and rapidly disassembled into mono-ubiquitin. We recently examined the toxicity and utilization of unanchored poly-ubiquitin in Drosophila melanogaster We found that free poly-ubiquitin species are largely innocuous to flies and that free poly-ubiquitin can be controlled by being degraded by the proteasome or by being conjugated onto another protein as a single unit. Here, to explore whether an organismal defense is mounted against unanchored chains, we conducted RNA-Seq analyses to examine the transcriptomic impact of free poly-ubiquitin in the fly. We found â¼90 transcripts whose expression is altered in the presence of different types of unanchored poly-ubiquitin. The set of genes identified was essentially devoid of ubiquitin-, proteasome-, or autophagy-related components. The seeming absence of a large and multipronged response to unanchored poly-ubiquitin supports the conclusion that these species need not be toxic in vivo and underscores the need to re-examine the role of free ubiquitin chains in the cell.
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Developability considerations should be integrated with lead engineering of antibody drug candidates in interest of their cost effective translations into medicines. To explore feasibility of this imperative, we have performed rational mutagenesis studies on a monoclonal antibody (MAB1) whose development was discontinued owing to manufacturability hurdles. Seven computationally designed variants of MAB1 containing single point (V44K, E59S, E59T and E59Y) and double (V44KE59S, V44KE59T and V44KE59Y) mutations in its light chain were produced in Chinese Hamster Ovary (CHO) cells and purified by using platform processes employed during commercial scale production of monoclonal antibodies. MAB1 and its variants were formulated in the same platform buffer and subjected to a battery of experiments to assess their solution behaviors, and biological activities. Five of the seven (71%) variants of MAB1 demonstrated improved biophysical attributes in multiple experimental testings. Contrary to the commonly expressed reservations about potential biological activity loss upon developability optimizations, the improvements in solution behavior of MAB1 also increased its biological activity up to ~180%. In particular, concentrate-ability and apparent solubility of V44KE59S improved to ~150% and ~160%, respectively. Its diffusion interaction parameter (kD) reduced to 28% and viscosity at ~100 mg/ml decreased to less than half of the corresponding values for MAB1. V44KE59S is also slightly more active and its transfections in CHO cells were more productive. It also degraded slower than MAB1 in three month long 25°C and 40°C formulation stability studies. These results open doors to an exciting realm of structure-based biologic drug design where developability and biological activity can be simultaneously optimized at the molecular engineering stages.
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Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Engenharia de Proteínas , Sequência de Aminoácidos , Anticorpos Monoclonais/genética , Linhagem Celular , Humanos , Simulação de Dinâmica Molecular , Mutação , Soluções , Temperatura , Termodinâmica , ViscosidadeRESUMO
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), the classic aryl hydrocarbon receptor (AhR) agonist, is a potent environmental toxicant and endocrine-disrupting chemical (EDC) with known developmental toxicity in humans, rodents, and fish. Early life exposure to some EDCs, including TCDD, is linked to the occurrence of adult-onset and multigenerational disease. Previous work exposing juvenile F0 zebrafish (Danio rerio) to 50 ppt (parts per trillion) TCDD during reproductive development has shown male-mediated transgenerational decreases in fertility (F0-F2) and histologic and transcriptomic alterations in F0 testes. Here, we analyzed male germline alterations in F1 and F2 adult fish, looking for changes in testicular histology and gene expression inherited through the male lineage that could account for decreased reproductive capacity. Testes of TCDD-lineage F1 fish displayed an increase in spermatogonia (immature germ cells) and decrease in spermatozoa (mature germ cells). No histological changes were present in F2 fish. Transcriptomic analysis of exposed F1 and F2 testes revealed alterations in lipid and glucose metabolism, oxidation, xenobiotic response, and sperm cell development and maintenance genes, all of which are implicated in fertility outcomes. Overall, we found that differential expression of reproductive genes and reduced capacity of sperm cells to mature could account for the reproductive defects previously seen in TCDD-exposed male zebrafish and their descendants, providing insight into the distinct multigenerational effects of toxicant exposure.
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Fertilidade/efeitos dos fármacos , Dibenzodioxinas Policloradas/toxicidade , Testículo/efeitos dos fármacos , Peixe-Zebra/genética , Animais , Substâncias Perigosas/toxicidade , Masculino , Reprodução/efeitos dos fármacos , Espermatogônias/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Transcriptoma/efeitos dos fármacosRESUMO
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental toxicant and endocrine disrupting compound with reproductive and developmental effects in humans and model organisms, including zebrafish. Our previous microarray and histological studies found defects in spermatogenesis and fertility of zebrafish in response to acute developmental TCDD exposure. These effects are apparent following exposure during reproductive development, modeling fetal basis of adult-onset disease. Some outcomes of these previous studies (reduced fertility, changes in sex ratio, transcriptomic alterations) are also transgenerational - persisting to unexposed generations - through the male germline. We hypothesized that DNA methylation could be a possible mechanism for these reproductive effects and performed whole genome bisulfite sequencing (WGBS), which identifies whole genome DNA methylation status at the base pair level, on testes of adult zebrafish exposed to TCDD (two separate hour-long exposures to 50 pg/mL TCDD at 3 and 7 weeks post fertilization). In response to TCDD exposure, multiple genes were differentially methylated; many of which are involved in reproductive processes or epigenetic modifications, suggesting a role of DNA methylation in later-life health outcomes. Additionally, several differentially methylated genes corresponded with gene expression changes identified in TCDD-exposed zebrafish testes, indicating a potential link between DNA methylation and gene expression. Ingenuity pathway analysis of WGBS and microarray data revealed genes involved in reproductive processes and development, RNA regulation, the cell cycle, and cellular morphology and development. We conclude that site-specific changes in DNA methylation of adult zebrafish testes occur in response to acute developmental TCDD exposure.
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We have shown that zebrafish (Danio rerio) are an excellent model for evaluating the link between early life stage exposure to environmental chemicals and disease in adulthood and subsequent unexposed generations. Previously, we used this model to identify transgenerational effects of dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin [TCDD]) on skeletal development, sex ratio, and reproductive capacity. Transgenerational inheritance of TCDD toxicity, notably decreased reproductive capacity, appears to be mediated through the male germ line. Thus, we examine testicular tissue for structural and gene expression changes using histology, microarray, and quantitative reverse transcription polymerase chain reaction (qRT-PCR). Histological analysis revealed decreased spermatozoa with concurrent increase in spermatogonia, and decreased germinal epithelium thickness in TCDD-exposed males compared with controls. We also identified altered expression of genes associated with testis development, steroidogenesis, spermatogenesis, hormone metabolism, and xenobiotic response. Altered genes are in pathways involving lipid metabolism, molecular transport, small molecule biochemistry, cell morphology, and metabolism of vitamins and minerals. These data will inform future investigations to elucidate the mechanism of adult-onset and transgenerational infertility due to TCDD exposure in zebrafish.