Exploratory analysis of transposable elements expression in the C. elegans early embryo

Background: Transposable Elements (TE) are mobile sequences that make up large portions of eukaryote genomes. The functions they play within the complex cellular architecture are still not clearly understood, but it is becoming evident that TE have a role in several physiological and pathological processes. In particular, it has been shown that TE transcription is necessary for the correct development of mice embryos and that their expression is able to finely modulate transcription of coding and non-coding genes. Moreover, their activity in the central nervous system (CNS) and other tissues has been correlated with the creation of somatic mosaicisms and with pathologies such as neurodevelopmental and neurodegenerative diseases as well as cancers. Results: We analyzed TE expression among different cell types of the Caenorhabditis elegans (C. elegans) early embryo asking if, where and when TE are expressed and whether their expression is correlated with genes playing a role in early embryo development. To answer these questions, we took advantage of a public C. elegans embryonic single-cell RNA-seq (sc-RNAseq) dataset and developed a bioinformatics pipeline able to quantify reads mapping specifically against TE, avoiding counting reads mapping on TE fragments embedded in coding/non-coding transcripts. Our results suggest that i) canonical TE expression analysis tools, which do not discard reads mapping on TE fragments embedded in annotated transcripts, may over-estimate TE expression levels, ii) Long Terminal Repeats (LTR) elements are mostly expressed in undifferentiated cells and might play a role in pluripotency maintenance and activation of the innate immune response, iii) non-LTR are expressed in differentiated cells, in particular in neurons and nervous system-Associated tissues, and iv) DNA TE are homogenously expressed throughout the C. elegans early embryo development. Conclusions: TE expression appears finely modulated in the C. elegans early embryo and different TE classes are expressed in different cell types and stages, suggesting that TE might play diverse functions during early embryo development

Changing trends of ocular trauma in the time of COVID-19 pandemic

To reduce the spread of the novel coronavirus (2019-nCoV), countries have promoted a range of unprecedented public health responses. These measures aim at reducing the final size of the epidemic as well as its peak in order to decrease the acute pressure on the health-care system [1]. In Italy, the government ordered people to stay home, restricting movements with the exception of work, urgent matters and health reasons. Furthermore, all commercial and productive activities, except those providing essential services, were obligated to remain closed [2]. Ocular trauma represents a serious public health problem and leading cause of visual impairment [3, 4]. The COVID-19 social distancing measures might have a significant impact on the risk of ocular trauma. In this study, we retrospectively reviewed the charts of all patients presenting to an Italian ophthalmological emergency department (the Ophthalmology Unit of the S.Orsola-Malpighi University Hospital in Bologna) to identify all eye injuries. Data were analysed from 10th March 2020 (i.e. the day in which the quarantine measures were applied in our city) to 10th April 2020, and confronted with those of the same period of the previous year (from 10th March 2019 to 10th April 2019). In the 2019 study period, there were 354 eye injuries (15.6% of all patients presenting to the emergency department). In the 2020 study period, eye injuries decreased to 112 (19.9% of all patients). The characteristics of eye injuries in the two study periods are reported in Table 1. During quarantine, the proportion of children and adolescents with eye injuries decreased (from 14.7% to 8.0%, Fig. 1a), while the proportion of males increased (from 66.7% to 75.0%, Fig. 1b). Regarding the mechanisms of injury, the percentage of falls and sport injuries had the highest decrease (respectively, from 6.5% to 0.9% and from 5.9% to 2.7%), while injuries during home activities and injuries with plants had the highest increase (respectively, from 12.4% to 17.0% and from 8.5% to 10.7%, Fig. 1c). The percentage of minor injuries with low risk of vision loss increased (from 93.2% to 94.6%), while major injuries requiring monitoring decreased (from 6.8% to 5.4%, Fig. 1d). There was a striking 68.4% decrease in the number of eye injuries seen in our Unit during the last month. Behavioural changes during the quarantine could be associated with lower risk of trauma. The decreases of sport injuries and of injuries in children during school closure seem to support this hypothesis. However, the drop of patients seeking emergency care affected all injuries, including serious ones potentially associated with vision loss. We believe that some patients may intentionally avoid urgent care rather than risking coronavirus exposure at hospitals. Anecdotal reports suggest that this is also happening for life-threatening medical emergencies such as myocardial infarction and stroke [5, 6]. Since ocular trauma is a major cause of vision loss, the importance of not delaying or avoiding treatment should be stressed to all patients to prevent ocular morbidities

Mimicking human riboflavin responsive neuromuscular disorders by silencing flad-1 gene in C. elegans: Alteration of vitamin transport and cholinergic transmission

Riboflavin (Rf), or vitamin B2, is the precursor of FMN and FAD, redox cofactors of several dehydrogenases involved in energy metabolism, redox balance and other cell regulatory processes. FAD synthase, coded by FLAD1 gene in humans, is the last enzyme in the pathway converting Rf into FAD. Mutations in FLAD1 gene are responsible for neuromuscular disorders, in some cases treatable with Rf. In order to mimic these disorders, the Caenorhabditis elegans (C. elegans) gene orthologue of FLAD1 (flad-1) was silenced in a model strain hypersensitive to RNA interference in nervous system. Silencing flad-1 resulted in a significant decrease in total flavin content, paralleled by a decrease in the level of the FAD-dependent ETFDH protein and by a secondary transcriptional down-regulation of the Rf transporter 1 (rft-1) possibly responsible for the total flavin content decrease. Conversely an increased ETFDH mRNA content was found. These biochemical changes were accompanied by significant phenotypical changes, including impairments of fertility and locomotion due to altered cholinergic transmission, as indicated by the increased sensitivity to aldicarb. A proposal is made that neuronal acetylcholine production/release is affected by alteration of Rf homeostasis. Rf supplementation restored flavin content, increased rft-1 transcript levels and eliminated locomotion defects. In this aspect, C. elegans could provide a low-cost animal model to elucidate the molecular rationale for Rf therapy in human Rf responsive neuromuscular disorders and to screen other molecules with therapeutic potential

Caenorhabditis elegans provides an efficient drug screening platform for GNAO1-related disorders and highlights the potential role of caffeine in controlling dyskinesia

Dominant GNAO1 mutations cause an emerging group of childhood-onset neurological disorders characterized by developmental delay, intellectual disability, movement disorders, drug-resistant seizures and neurological deterioration. GNAO1 encodes the α-subunit of an inhibitory GTP/GDP-binding protein regulating ion channel activity and neurotransmitter release. The pathogenic mechanisms underlying GNAO1-related disorders remain largely elusive and there are no effective therapies. Here, we assessed the functional impact of two disease-causing variants associated with distinct clinical features, c.139A > G (p.S47G) and c.662C > A (p.A221D), using Caenorhabditis elegans as a model organism. The c.139A > G change was introduced into the orthologous position of the C. elegans gene via CRISPR/Cas9, whereas a knock-in strain carrying the p.A221D variant was already available. Like null mutants, homozygous knock-in animals showed increased egg laying and were hypersensitive to aldicarb, an inhibitor of acetylcholinesterase, suggesting excessive neurotransmitter release by different classes of motor neurons. Automated analysis of C. elegans locomotion indicated that goa-1 mutants move faster than control animals, with more frequent body bends and a higher reversal rate and display uncoordinated locomotion. Phenotypic profiling of heterozygous animals revealed a strong hypomorphic effect of both variants, with a partial dominant-negative activity for the p.A221D allele. Finally, caffeine was shown to rescue aberrant motor function in C. elegans harboring the goa-1 variants; this effect is mainly exerted through adenosine receptor antagonism. Overall, our findings establish a suitable platform for drug discovery, which may assist in accelerating the development of new therapies for this devastating condition, and highlight the potential role of caffeine in controlling GNAO1-related dyskinesia

Glucose-Coated Superparamagnetic Iron Oxide Nanoparticles Prepared by Metal Vapour Synthesis Are Electively Internalized in a Pancreatic Adenocarcinoma Cell Line Expressing GLUT1 Transporter

Iron oxide nanoparticles (IONP) can have a variety of biomedical applications due to their visualization properties through Magnetic Resonance Imaging (MRI) and heating with radio frequency or alternating magnetic fields. In the oncological field, coating IONP with organic compounds to provide specific features and to achieve the ability of binding specific molecular targets appears to be very promising. To take advantage of the high avidity of tumor cells for glucose, we report the development of very small glucose-coated IONP (glc-IONP) by employing an innovative technique, Metal Vapor Synthesis (MVS). Moreover, we tested the internalization of our gl-IONP on a tumor line, BxPC3, over-expressing GLUT 1 transporter. Both glc-IONP and polyvinylpyrrolidone-IONP (PVP-IONP), as control, were prepared with MVS and were tested on BxPC3 at various concentrations. To evaluate the role of GLUT-1 transporter, we also investigated the effect of adding a polyclonal anti-GLUT1 antibody. After proper treatment, the iron value was assessed by atomic absorption spectrometer, reported in mcg/L and expressed in mg of protein. Our IONP prepared with MVS were very small and homogeneously distributed in a narrow range (1.75-3.75 nm) with an average size of 2.7 nm and were super-paramagnetic. Glc-IONP were internalized by BxPC3 cells in a larger amount than PVP-IONP. After 6h of treatment with 50 mcg/mL of IONPs, the content of Fe was 1.5 times higher in glc-IONP-treated cells compared with PVP-IONP-treated cells. After 1h pre-treatment with anti-GLUT1, a reduction of 41% cellular accumulation of glc-IONP was observed. Conversely, the uptake of PVP-IONPs was reduced only by 14% with antibody pretreatment. In conclusion, MVS allowed us to prepare small, homogeneous, super-paramagnetic glc-IONP, which are electively internalized by a tumor line over-expressing GLUT1. Our glc-IONP appear to have many requisites for in vivo use

Irradiation detection of herbal ingredients used in plant food supplements by Electron Spin Resonance on samples pre-treated with alcoholic extraction

This study aimed to verify the applicability of the EN 1787 method for the detection of irradiation in herbal ingredients used in Plant Food Supplements (PFSs). In matrices such as herbs and spices the main limit of the method is the presence of intrinsic radicals responsible for spurious signals leading to complex ESR spectra. To overcome this limit, before ESR measurement a treatment with alcohol has been proposed (Delincée and Soika, 2002; Ahn et al., 2012, 2014). As reported in the literature, this treatment is expected to reduce/eliminate the confounding signals so that the samples may be correctly classified. In this study the efficacy of the pre-treatment was tested on raw herbal ingredients largely used for PFSs, namely Camellia sinensis, Cinnamomum verum, Curcuma longa, Ginkgo biloba, Silybum marianum, Vaccinium myrtillus and Zingiber officinale. Non-irradiated and irradiated (5, 10 kGy) samples were analysed before and after pre-treatment. The results showed a general decrement of signal intensity. In some cases, this was associated with the elimination of some spurious signals, which, however, did not always ensue in an easier interpretation of the ESR spectra. Only for two matrices (Camellia sinensis and Vaccinium myrtillus) was alcoholic extraction crucial for the correct classification of the samples

An inter-laboratory comparison to evaluate the suitability of EN 1787 standard to detect irradiation in plant-origin foods with health benefits

This paper reports the results of a study carried out to verify the applicability of the EN 1787 method, which uses the Electron Spin Resonance (ESR) technique for the identification of irradiated plant-origin foods with health benefits. The method was tested on samples of herbal ingredients of Plant Food Supplements (PFSs), nuts and fresh blueberries. Untreated and irradiated samples of Camellia sinensis (leaves) Ginkgo biloba (leaves), Glycine max (seeds), Silybum marianum (fruits), Vaccinium myrtillus (fruits), almonds, hazelnuts, peanuts, pistachios, walnuts and fresh blueberries were analysed. The work includes an inter-laboratory blind test involving five Italian laboratories that perform routine analyses for the official control of irradiated food. A total of 180 untreated and irradiated samples of PFS ingredients, nuts and fresh blueberries were analysed. The analyses on the irradiated samples were replicated even a long time after irradiation (up to two years depending on the matrix) to test the reliability of the method throughout the shelf life of the products. The results were matrix-dependent: all the 5 kGy irradiated nuts and the 1 kGy-irradiated blueberries were correctly classified, whereas herbal ingredients showed complex ESR spectra with spurious signals which often prevented the correct classification of the sample

C. elegans expressing D76N β2-microglobulin: a model for in vivo screening of drug candidates targeting amyloidosis

The availability of a genetic model organism with which to study key molecular events underlying amyloidogenesis is crucial for elucidating the mechanism of the disease and the exploration of new therapeutic avenues. The natural human variant of β2-microglobulin (D76N β2-m) is associated with a fatal familial form of systemic amyloidosis. Hitherto, no animal model has been available for studying in vivo the pathogenicity of this protein. We have established a transgenic C. elegans line, expressing the human D76N β2-m variant. Using the INVertebrate Automated Phenotyping Platform (INVAPP) and the algorithm Paragon, we were able to detect growth and motility impairment in D76N β2-m expressing worms. We also demonstrated the specificity of the β2-m variant in determining the pathological phenotype by rescuing the wild type phenotype when β2-m expression was inhibited by RNA interference (RNAi). Using this model, we have confirmed the efficacy of doxycycline, an inhibitor of the aggregation of amyloidogenic proteins, in rescuing the phenotype. In future, this C. elegans model, in conjunction with the INVAPP/Paragon system, offers the prospect of high-throughput chemical screening in the search for new drug candidates