Is Cadmium Toxicity Tissue-Specific? Toxicogenomics Studies Reveal Common and Specific Pathways in Pulmonary, Hepatic, and Neuronal Cell Models.

Several harmful modifications in different tissues-organs, leading to relevant diseases (e.g., liver and lung diseases, neurodegeneration) are reported after exposure to cadmium (Cd), a wide environmental contaminant. This arises the question whether any common molecular signatures and/or Cd-induced modifications might represent the building block in initiating or contributing to address the cells towards different pathological conditions. To unravel possible mechanisms of Cd tissue-specificity, we have analyzed transcriptomics data from cell models representative of three major Cd targets: pulmonary (A549), hepatic (HepG2), and neuronal (SH-SY-5Y) cells. Further, we compared common features to identify any non-specific molecular signatures. The functional analysis of dysregulated genes (gene ontology and KEGG) shows GO terms related to metabolic processes significantly enriched only in HepG2 cells. GO terms in common in the three cell models are related to metal ions stress response and detoxification processes. Results from KEGG analysis show that only one specific pathway is dysregulated in a significant way in all cell models: the mineral absorption pathway. Our data clearly indicate how the molecular mimicry of Cd and its ability to cause a general metal ions dyshomeostasis represent the initial common feature leading to different molecular signatures and alterations, possibly responsible for different pathological conditions.

Analyses of Transcriptomics Cell Signalling for Pre-Screening Applications in the Integrated Approach for Testing and Assessment of Non-Genotoxic Carcinogens.

With recent rapid advancement of methodological tools, mechanistic understanding of biological processes leading to carcinogenesis is expanding. New approach methodologies such as transcriptomics can inform on non-genotoxic mechanisms of chemical carcinogens and can be developed for regulatory applications. The Organisation for the Economic Cooperation and Development (OECD) expert group developing an Integrated Approach to the Testing and Assessment (IATA) of Non-Genotoxic Carcinogens (NGTxC) is reviewing the possible assays to be integrated therein. In this context, we review the application of transcriptomics approaches suitable for pre-screening gene expression changes associated with phenotypic alterations that underlie the carcinogenic processes for subsequent prioritisation of downstream test methods appropriate to specific key events of non-genotoxic carcinogenesis. Using case studies, we evaluate the potential of gene expression analyses especially in relation to breast cancer, to identify the most relevant approaches that could be utilised as (pre-) screening tools, for example Gene Set Enrichment Analysis (GSEA). We also consider how to address the challenges to integrate gene panels and transcriptomic assays into the IATA, highlighting the pivotal omics markers identified for assay measurement in the IATA key events of inflammation, immune response, mitogenic signalling and cell injury.

miRandb: a resource of online services for miRNA research.

Recent discovery of thousands of small and large noncoding RNAs, in parallel to technical improvements enabling scientists to study the transcriptome in much higher depth, has resulted in massive data generation. This burst of information prompts the development of easily accessible resources for storage, retrieval and analysis of raw and processed data, and hundreds of Web-based tools dedicated to these tasks have been made available. However, the increasing number and diversity of bioinformatics tools, each covering a specific and specialized area, as well as their redundancies, represent potential sources of complication for end users. To overcome these issues, we are introducing an easy-to-follow classification of microRNA (miRNA)-related bioinformatics tools for biologists interested in studying this important class of small noncoding RNAs. We also developed our miRNA database miRNA algorithmic network database (miRandb) that is a meta-database, which presents a survey of > 180 Web-based miRNA databases. These include miRNA sequence, discovery, target prediction, target validation, expression and regulation, functions and their roles in diseases, interactions in cellular pathways and networks and deep sequencing. miRandb recapitulates the diverse possibilities and facilitates that access to the different categories of miRNA resources. Researchers can easily select the category of miRNA information and desired organism, in result eligible databases with their features are presented. This database introducing an easy-to-follow classification of available resources that can facilitate selection of appropriate resources for miRNA-related bioinformatics tools. Finally, we described current shortages and future necessities that assist researchers to use these tools easily. Our database is accessible at http://mirandb.ir.

Rotenone exerts developmental neurotoxicity in a human brain spheroid model.

Growing concern suggests that some chemicals exert (developmental) neurotoxicity (DNT and NT) and are linked to the increase in incidence of autism, attention deficit and hyperactivity disorders. The high cost of routine tests for DNT and NT assessment make it difficult to test the high numbers of existing chemicals. Thus, more cost effective neurodevelopmental models are needed. The use of induced pluripotent stem cells (iPSC) in combination with the emerging human 3D tissue culture platforms, present a novel tool to predict and study human toxicity. By combining these technologies, we generated multicellular brain spheroids (BrainSpheres) from human iPSC. The model has previously shown to be reproducible and recapitulates several neurodevelopmental features. Our results indicate, rotenone's toxic potency varies depending on the differentiation status of the cells, showing higher reactive oxygen species (ROS) and higher mitochondrial dysfunction during early than later differentiation stages. Immuno-fluorescence morphology analysis after rotenone exposure indicated dopaminergic-neuron selective toxicity at non-cytotoxic concentrations (1 µM), while astrocytes and other neuronal cell types were affected at (general) cytotoxic concentrations (25 µM). Omics analysis showed changes in key pathways necessary for brain development, indicating rotenone as a developmental neurotoxicant and show a possible link between previously shown effects on neurite outgrowth and presently observed effects on Ca2+ reabsorption, synaptogenesis and PPAR pathway disruption. In conclusion, our BrainSpheres model has shown to be a reproducible and novel tool to study neurotoxicity and developmental neurotoxicity. Results presented here support the idea that rotenone can potentially be a developmental neurotoxicant.

Cell-free nucleic acids are present in blood products and regulate genes of innate immune response.

BACKGROUND: Extracellular nucleic acids circulate in plasma. They are expected to be present in manufactured blood products eligible for transfusion, but little is known about their biological activity on human cells. The aim of this study is to investigate whether cell-free nucleic acids (CFNAs) are present and biologically active in red blood cell units (RBCUs), fresh frozen plasmas, and platelet concentrates. STUDY DESIGN AND METHODS: CFNAs were extracted from RBCUs, fresh frozen plasma, and platelet concentrates. Their nature and structure were analyzed by regular methods of nucleic acid detection/quantification. A normalized polymerase chain reaction combining amplification of a CFNA marker (Alu 115) and amplification of an internal nonhuman DNA control spiked in all samples (phiX 174) was developed to study CFNA release after RBCU storage. The impact of CFNAs on gene regulation was tested by microarray after coculture with peripheral blood mononuclear cells and macrophages. RESULTS: Extracellular double-stranded DNA was present in all blood products, with higher amounts found in cellular suspensions (RBCUs and platelet concentrates). Storage up to 40 days did not influence release from RBCUs, and CFNA amount varied considerably from one unit to another. Microarray experiments showed that exposition of macrophages to CFNA increased the expression of genes involved in the innate immune response including chemokines, chemokine receptors, and receptors of the innate response. CONCLUSION: CFNAs are present in blood products. Immunoregulatory properties of CFNA are shown in vitro, providing new insights on biologically active components of blood products besides those for intended therapeutic use.

Metagenomics analysis of red blood cell and fresh-frozen plasma units.

BACKGROUND: Although the risk of transmitting infectious agents by blood transfusion is dramatically reduced after donor selection, leukoreduction, and laboratory testing, some could still be present in donor's blood. A description of metagenomes in blood products eligible for transfusion represents relevant information to evaluate the risk of pathogen transmission by transfusion. STUDY DESIGN AND METHODS: Detection of viruses, bacteria, and fungi genomes was made by high-throughput sequencing (HTS) of 600 manufactured blood products eligible for transfusion: 300 red blood cell (RBC) and 300 fresh-frozen plasma (FFP) units. RESULTS: Anelloviruses and human pegivirus, frequent in the blood of healthy individuals, were found. Human papillomavirus type 27 and Merkel cell polyomavirus, present on the skin, were also detected. Unexpectedly, astrovirus MLB2 was identified and characterized in a FFP unit. The presence of astrovirus MLB2 was confirmed in donor's blood and corresponded to an asymptomatic acute viremia. Sequences of bacteria and fungi were also detected; they are likely the result of environmental contamination. CONCLUSION: This study demonstrates that HTS is a promising tool for detecting common and less frequent infectious pathogens in blood products.

Salt preferences of honey bee water foragers.

The importance of dietary salt may explain why bees are often observed collecting brackish water, a habit that may expose them to harmful xenobiotics. However, the individual salt preferences of water-collecting bees were not known. We measured the proboscis extension reflex (PER) response of Apis mellifera water foragers to 0-10% w/w solutions of Na, Mg and K, ions that provide essential nutrients. We also tested phosphate, which can deter foraging. Bees exhibited significant preferences, with the most PER responses for 1.5-3% Na and 1.5% Mg. However, K and phosphate were largely aversive and elicited PER responses only for the lowest concentrations, suggesting a way to deter bees from visiting contaminated water. We then analyzed the salt content of water sources that bees collected in urban and semi-urban environments. Bees collected water with a wide range of salt concentrations, but most collected water sources had relatively low salt concentrations, with the exception of seawater and swimming pools, which had >0.6% Na. The high levels of PER responsiveness elicited by 1.5-3% Na may explain why bees are willing to collect such salty water. Interestingly, bees exhibited high individual variation in salt preferences: individual identity accounted for 32% of variation in PER responses. Salt specialization may therefore occur in water foragers.

Deficiency of the miR-29a/b-1 cluster leads to ataxic features and cerebellar alterations in mice.

miR-29 is expressed strongly in the brain and alterations in expression have been linked to several neurological disorders. To further explore the function of this miRNA in the brain, we generated miR-29a/b-1 knockout animals. Knockout mice develop a progressive disorder characterized by locomotor impairment and ataxia. The different members of the miR-29 family are strongly expressed in neurons of the olfactory bulb, the hippocampus and in the Purkinje cells of the cerebellum. Morphological analysis showed that Purkinje cells are smaller and display less dendritic arborisation compared to their wildtype littermates. In addition, a decreased number of parallel fibers form synapses on the Purkinje cells. We identified several mRNAs significantly up-regulated in the absence of the miR-29a/b-1 cluster. At the protein level, however, the voltage-gated potassium channel Kcnc3 (Kv3.3) was significantly up-regulated in the cerebella of the miR-29a/b knockout mice. Dysregulation of KCNC3 expression may contribute to the ataxic phenotype.

APLP2 regulates neuronal stem cell differentiation during cortical development.

Expression of amyloid precursor protein (APP) and its two paralogues, APLP1 and APLP2 during brain development coincides with key cellular events such as neuronal differentiation and migration. However, genetic knockout and shRNA studies have led to contradictory conclusions about their role during embryonic brain development. To address this issue, we analysed in depth the role of APLP2 during neurogenesis by silencing APLP2 in vivo in an APP/APLP1 double knockout mouse background. We find that under these conditions cortical progenitors remain in their undifferentiated state much longer, displaying a higher number of mitotic cells. In addition, we show that neuron-specific APLP2 downregulation does not impact the speed or position of migrating excitatory cortical neurons. In summary, our data reveal that APLP2 is specifically required for proper cell cycle exit of neuronal progenitors, and thus has a distinct role in priming cortical progenitors for neuronal differentiation.

On the identification of low allele frequency mosaic mutations in the brains of Alzheimer's disease patients.

INTRODUCTION: The cause of sporadic Alzheimer's disease (AD) remains unclear. Given the growing evidence that protein aggregates can spread in a "prion-like" fashion, we reasoned that a small population of brain cells producing such "prion-like" particles due to a postzygotic acquired mutation would be sufficient to trigger the disease. Deep DNA sequencing technology should in principle allow the detection of such mosaics. METHODS: To detect the somatic mutations of genes causing AD present in a small number of cells, we developed a targeted deep sequencing approach to scrutinize the genomic loci of APP, PSEN1, and PSEN2 genes in DNA extracted from the entorhinal cortex, one of the brain regions showing the earliest signs of AD pathology. We also included the analysis of the MAPT gene because mutations may promote tangle formation. We validated candidate mutations with an independent targeted ultradeep amplicon sequencing technique. RESULTS: We demonstrate that our approach can detect single-nucleotide mosaic variants with a 1% allele frequency and copy number mosaic variants present in as few as 10% of cells. We screened 72 AD and 58 control brain samples and identified three mosaic variants with low allelic frequency (∼1%): two novel MAPT variants in sporadic AD patients and a known PSEN2 variant in a Braak II control subject. Moreover, we detected both novel and known pathogenic nonmosaic heterozygous variants in PSEN1 and PSEN2 in this cohort of sporadic AD patients. DISCUSSION: Our results show that mosaic mutations with low allelic frequencies in AD-relevant genes can be detected in brain-derived DNA, but larger samples need to be investigated before a more definitive conclusion with regard to the pathogenicity of such mosaics can be made.

Sex-biasing influence of autism-associated Ube3a gene overdosage at connectomic, behavioral, and transcriptomic levels.

Genomic mechanisms enhancing risk in males may contribute to sex bias in autism. The ubiquitin protein ligase E3A gene (Ube3a) affects cellular homeostasis via control of protein turnover and by acting as transcriptional coactivator with steroid hormone receptors. Overdosage of Ube3a via duplication or triplication of chromosomal region 15q11-13 causes 1 to 2% of autistic cases. Here, we test the hypothesis that increased dosage of Ube3a may influence autism-relevant phenotypes in a sex-biased manner. We show that mice with extra copies of Ube3a exhibit sex-biasing effects on brain connectomics and autism-relevant behaviors. These effects are associated with transcriptional dysregulation of autism-associated genes, as well as genes differentially expressed in 15q duplication and in autistic people. Increased Ube3a dosage also affects expression of genes on the X chromosome, genes influenced by sex steroid hormone, and genes sex-differentially regulated by transcription factors. These results suggest that Ube3a overdosage can contribute to sex bias in neurodevelopmental conditions via influence on sex-differential mechanisms.

MicroRNAs in the pineal gland: miR-483 regulates melatonin synthesis by targeting arylalkylamine N-acetyltransferase.

MicroRNAs (miRNAs) play a broad range of roles in biological regulation. In this study, rat pineal miRNAs were profiled for the first time, and their importance was evaluated by focusing on the main function of the pineal gland, melatonin synthesis. Massively parallel sequencing and related methods revealed the miRNA population is dominated by a small group of miRNAs as follows: ~75% is accounted for by 15 miRNAs; miR-182 represents 28%. In addition to miR-182, miR-183 and miR-96 are also highly enriched in the pineal gland, a distinctive pattern also found in the retina. This effort also identified previously unrecognized miRNAs and other small noncoding RNAs. Pineal miRNAs do not exhibit a marked night/day difference in abundance with few exceptions (e.g. 2-fold night/day differences in the abundance of miR-96 and miR-182); this contrasts sharply with the dynamic 24-h pattern that characterizes the pineal transcriptome. During development, the abundance of most pineal gland-enriched miRNAs increases; however, there is a marked decrease in at least one, miR-483. miR-483 is a likely regulator of melatonin synthesis, based on the following. It inhibits melatonin synthesis by pinealocytes in culture; it acts via predicted binding sites in the 3"-UTR of arylalkylamine N-acetyltransferase (Aanat) mRNA, the penultimate enzyme in melatonin synthesis, and it exhibits a developmental profile opposite to that of Aanat transcripts. Additionally, a miR-483 targeted antagonist increased melatonin synthesis in neonatal pinealocytes. These observations support the hypothesis that miR-483 suppresses Aanat mRNA levels during development and that the developmental decrease in miR-483 abundance promotes melatonin synthesis.

The microbiome and gene expression of honey bee workers are affected by a diet containing pollen substitutes.

Pollen is the primary source of dietary protein for honey bees. It also includes complex polysaccharides in its outer coat, which are largely indigestible by bees but can be metabolized by bacterial species within the gut microbiota. During periods of reduced availability of floral pollen, supplemental protein sources are frequently provided to managed honey bee colonies. The crude proteins in these supplemental feeds are typically byproducts from food manufacturing processes and are rarely derived from pollen. Our experiments on the impact of different diets showed that a simplified pollen-free diet formulated to resemble the macronutrient profile of a monofloral pollen source resulted in larger microbial communities with reduced diversity, reduced evenness, and reduced levels of potentially beneficial hive-associated bacteria. Furthermore, the pollen-free diet sharply reduced the expression of genes central to honey bee development. In subsequent experiments, we showed that these shifts in gene expression may be linked to colonization by the gut microbiome. Lastly, we demonstrated that for bees inoculated with a defined gut microbiota, those raised on an artificial diet were less able to suppress infection from a bacterial pathogen than those that were fed natural pollen. Our findings demonstrate that a pollen-free diet significantly impacts the gut microbiota and gene expression of honey bees, indicating the importance of natural pollen as a primary protein source.

LINE-1 regulates cortical development by acting as long non-coding RNAs.

Long Interspersed Nuclear Elements-1s (L1s) are transposable elements that constitute most of the genome's transcriptional output yet have still largely unknown functions. Here we show that L1s are required for proper mouse brain corticogenesis operating as regulatory long non-coding RNAs. They contribute to the regulation of the balance between neuronal progenitors and differentiation, the migration of post-mitotic neurons and the proportions of different cell types. In cortical cultured neurons, L1 RNAs are mainly associated to chromatin and interact with the Polycomb Repressive Complex 2 (PRC2) protein subunits enhancer of Zeste homolog 2 (Ezh2) and suppressor of zeste 12 (Suz12). L1 RNA silencing influences PRC2's ability to bind a portion of its targets and the deposition of tri-methylated histone H3 (H3K27me3) marks. Our results position L1 RNAs as crucial signalling hubs for genome-wide chromatin remodelling, enabling the fine-tuning of gene expression during brain development and evolution.

Dysregulated microRNAs in neurodegenerative disorders.

The complexity of the nervous system arises in part, from the large diversity of neural cell types that support the architecture of neuronal circuits. Recent studies have highlighted microRNAs as important players in regulating gene expression at the post-transcriptional level and therefore the phenotype of neural cells. A link between microRNAs and neurodegenerative diseases such as Alzheimer's disease, Huntington's disease and Parkinson's disease is becoming increasingly evident. Here, we discuss microRNAs in neurodegeneration, from the fruit fly and mouse utilized as experimental models to dysregulated microRNAs in human neurodegenerative disorders. We propose that studying microRNAs and their mRNA targets in the context of neurodegeneration will significantly contribute to the identification of proteins important for neuronal function and might reveal underlying molecular networks that drive these diseases.

Assessing pollen nutrient content: a unifying approach for the study of bee nutritional ecology.

Poor nutrition and landscape changes are regularly cited as key factors causing the decline of wild and managed bee populations. However, what constitutes 'poor nutrition' for bees currently is inadequately defined. Bees collect and eat pollen: it is their only solid food source and it provides a broad suite of required macro- and micronutrients. Bees are also generalist foragers and thus the different pollen types they collect and eat can be highly nutritionally variable. Therefore, characterizing the multidimensional nutrient content of different pollen types is needed to fully understand pollen as a nutritional resource. Unfortunately, the use of different analytical approaches to assess pollen nutrient content has complicated between-studies comparisons and blurred our understanding of pollen nutrient content. In the current study, we start by reviewing the common methods used to estimate protein and lipids found in pollen. Next, using monofloral Brassica and Rosa pollen, we experimentally reveal biases in results using these methods. Finally, we use our collective data to propose a unifying approach for analysing pollen nutrient content. This will help researchers better study and understand the nutritional ecology-including foraging behaviour, nutrient regulation and health-of bees and other pollen feeders. This article is part of the theme issue 'Natural processes influencing pollinator health: from chemistry to landscapes'.

Honey Bee (Apis mellifera) Exposure to Pesticide Residues in Nectar and Pollen in Urban and Suburban Environments from Four Regions of the United States.

The risk of honey bee (Apis mellifera L.) exposure to pesticide residues while foraging for nectar and pollen is commonly explored in the context of agroecosystems. However, pesticides are also used in urban and suburban areas for vegetation management, vector control, and the management of ornamental plants in public and private landscapes. The extent to which pesticides pose a health risk to honey bees in these settings remains unclear. We addressed this at a landscape scale by conducting pesticide residue screening analyses on 768 nectar and 862 pollen samples collected monthly over 2 years from honey bee colonies located in urban and suburban areas in eight medium to large cities in California, Florida, Michigan, and Texas (USA). A risk assessment was performed using the US Environmental Protection Agency's BeeREX model whenever an oral toxicity value was available for a compound. Chemical analyses detected 17 pesticides in nectar and 60 in pollen samples during the survey. Approximately 73% of all samples contained no detectable pesticide residues. Although the number of detections varied among the sampled regions, fewer pesticides were detected in nectar than in pollen. Per BeeREX, four insecticides showed a potential acute risk to honey bees: imidacloprid, chlorpyrifos, and esfenvalerate in nectar, and deltamethrin in nectar and pollen. In general, exposure of honey bees to pesticides via nectar and pollen collection was low in urban and suburban areas across the United States, and no seasonal or spatial trends were evident. Our data suggest that honey bees are exposed to fewer pesticides in developed areas than in agricultural ones. Environ Toxicol Chem 2022;41:991-1003. © 2022 SETAC.

TPPP/p25 promotes tubulin acetylation by inhibiting histone deacetylase 6.

TPPP/p25 (tubulin polymerization-promoting protein/p25) is an unstructured protein that induces microtubule polymerization in vitro and is aligned along the microtubule network in transfected mammalian cells. In normal human brain, TPPP/p25 is expressed predominantly in oligodendrocytes, where its expression is proved to be crucial for their differentiation process. Here we demonstrated that the expression of TPPP/p25 in HeLa cells, in doxycycline-inducible CHO10 cells, and in the oligodendrocyte CG-4 cells promoted the acetylation of alpha-tubulin at residue Lys-40, whereas its down-regulation by specific small interfering RNA in CG-4 cells or by the withdrawal of doxycycline from CHO10 cells decreased the acetylation level of alpha-tubulin. Our results indicate that TPPP/p25 binds to HDAC6 (histone deacetylase 6), an enzyme responsible for tubulin deacetylation. Moreover, we demonstrated that the direct interaction of these two proteins resulted in the inhibition of the deacetylase activity of HDAC6. The measurement of HDAC6 activity showed that TPPP/p25 is able to induce almost complete (90%) inhibition at 3 microM concentration. In addition, treatment of the cells with nocodazole, vinblastine, or cold exposure revealed that microtubule acetylation induced by trichostatin A, a well known HDAC6 inhibitor, does not cause microtubule stabilization. In contrast, the microtubule bundling activity of TPPP/p25 was able to protect the microtubules from depolymerization. Finally, we demonstrated that, similarly to other HDAC6 inhibitors, TPPP/p25 influences the microtubule dynamics by decreasing the growth velocity of the microtubule plus ends and also affects cell motility as demonstrated by time lapse video experiments. Thus, we suggest that TPPP/p25 is a multiple effector of the microtubule organization.

Tubulin polymerization-promoting protein (TPPP/p25) is critical for oligodendrocyte differentiation.

TPPP/p25, a recently identified tubulin polymerization-promoting protein (TPPP), is expressed mainly in myelinating oligodendrocytes of the CNS. Here, we show that TPPP/p25 is strongly upregulated during the differentiation of primary oligodendrocyte cells as well as the CG-4 cell line. The microRNA expression profile of CG-4 cells before and after induction of differentiation was established and revealed differential regulation of a limited subset of microRNAs. miR-206, a microRNA predicted to target TPPP/p25, was not detected in oligodendrocytes. Overexpression of miR-206 led to downregulation of TPPP/p25 resulting in inhibition of differentiation. Transfection of siRNAs against TPPP/p25 also inhibited cell differentiation and promoted cell proliferation, providing evidence for an important role of TPPP/p25 during oligodendrogenesis. These results support an essential role for TPPP/p25 in oligodendrocyte differentiation likely via rearrangement of the microtubule system during the process elongation prior to the onset of myelination.