Search

Two analogues of the MS3 aptamer, which was previously shown to have an exquisite capability to selectively bind and modulate the activity of mutant huntingtin (mHTT), have been here designed and evaluated in their physicochemical and biological properties. Featured by a distinctive propensity to form complex G-quadruplex structures, including large multimeric aggregates, the original 36-mer MS3 has been truncated to give a 33-mer (here named MS3-33) and a 17-mer (here named MS3-17). A combined use of different techniques (UV, CD, DSC, gel electrophoresis) allowed a detailed physicochemical characterization of these novel G-quadruplex-forming aptamers, tested in vitro on SH-SY5Y cells and in vivo on a Drosophila Huntington’s disease model, in which these shorter MS3-derived oligonucleotides proved to have improved bioactivity in comparison with the parent aptamer.

A set of guanine-rich aptamers able to preferentially recognize full-length huntingtin with an expanded polyglutamine tract has been recently identified, showing high efficacy in modulating the functions of the mutated protein in a variety of cell experiments. We here report a detailed biophysical characterization of the best aptamer in the series, named MS3, proved to adopt a stable, parallel G-quadruplex structure and show high nuclease resistance in serum. Confocal microscopy experiments on HeLa and SH-SY5Y cells, as models of non-neuronal and neuronal cells, respectively, showed a rapid, dose-dependent uptake of fluorescein-labelled MS3, demonstrating its effective internalization, even in the absence of transfecting agents, with no general cytotoxicity. Then, using a well-established Drosophila melanogaster model for Huntington’s disease, which expresses the mutated form of human huntingtin, a significant improvement in the motor neuronal function in flies fed with MS3 was observed, proving the in vivo efficacy of this aptamer.

A transition from one developmental stage to another is accompanied by activation of developmental programs and corresponding gene ensembles. Changes in the spatial conformation of the corresponding loci are associated with this activation and can be investigated with the help of the Chromosome Conformation Capture (3C) methodology. Application of 3C to specific developmental stages is a sophisticated task. Here, we describe the use of the 3C method to study the spatial organization of developmental loci in

The chromatin remodeler SWI/SNF is an important participant in gene activation, functioning predominantly by opening the chromatin structure on promoters and enhancers. Here, we describe its novel mode of action in which SWI/SNF factors mediate the targeted action of an enhancer. We studied the functions of two signature subunits of PBAP subfamily, BAP170 and SAYP, in Drosophila. These subunits were stably tethered to a transgene reporter carrying the hsp70 core promoter. The tethered subunits mediate transcription of the reporter in a pattern that is generated by enhancers close to the insertion site in multiple loci throughout the genome. Both tethered SAYP and BAP170 recruit the whole PBAP complex to the reporter promoter. However, we found that BAP170-dependent transcription is more resistant to the depletion of other PBAP subunits, suggesting that BAP170 may play a more critical role in establishing enhancer-dependent transcription.

A mismatch between &beta, oxidation and the tricarboxylic acid cycle (TCA) cycle flux in mitochondria produces an accumulation of lipid metabolic intermediates, resulting in both blunted metabolic flexibility and decreased glucose utilization in the affected cells. The ability of the cell to switch to glucose as an energy substrate can be restored by reducing the reliance of the cell on fatty acid oxidation. The inhibition of the carnitine system, limiting the carnitine shuttle to the oxidation of lipids in the mitochondria, allows cells to develop a high plasticity to metabolic rewiring with a decrease in fatty acid oxidation and a parallel increase in glucose oxidation. We found that 3-(2,2,2-trimethylhydrazine)propionate (THP), which is able to reduce cellular carnitine levels by blocking both carnitine biosynthesis and the cell membrane carnitine/organic cation transporter (OCTN2), was reported to improve mitochondrial dysfunction in several diseases, such as Huntington&rsquo, s disease (HD). Here, new THP-derived carnitine-lowering agents (TCL), characterized by a high affinity for the OCTN2 with a minimal effect on carnitine synthesis, were developed, and their biological activities were evaluated in both in vitro and in vivo HD models. Certain compounds showed promising biological activities: reducing protein aggregates in HD cells, ameliorating motility defects, and increasing the lifespan of HD Drosophila melanogaster.

17-a-ethinylestradiol (EE2) belongs to the increasing list of Endocrine Disruptors Chemicals (EDCs), able to interfere with the endocrine system in both vertebrates and invertebrates. Regardless of its great dispersion in the environment, to date there is still little knowledge about its action mechanisms and harmful effects in invertebrates. To better evaluate its potential role in invertebrates, we used the model system Drosophila melanogaster, an insect in which the hormonal response has been widely described. The effects of EE2 in D.melanogaster adults have been evaluated by using life traits as well as molecular endpoints. It was found that EE2 significantly decreases survival and fertility in both sexes, with a higher effect in female flies, as well as affects the expression of the Ecdysone Receptor (EcR), Estrogen Related Receptor (ERR), Yolk protein2 (Yp2) and yolkless (yl) genes. In conclusion, our results suggest that EE2 treatment may have potential toxic and endocrine effects on Drosophila melanogaster adults of both sexes. In particular, our data provide an indication that, after EE2 treatment, two of the genes involved in the vitellogenesis process (yl and Yp2) are transcribed in adult males where are mostly silent, and suggest future studies forward their use as potential molecular markers to EDCs exposure in Drosophila male.

Nowadays, Quality Management tools such as failure mode and effect analysis (FMEA) are widely used throughout the aeronautical, automotive, software, food services, health care and many other industries to sustain and improve quality and safety. The increasing complexity of scientific research makes it more difficult to maintain all activities under control, in order to guarantee validity and reproducibility of results. Even in non-regulated research, scientists need to be supported with management tools that maximize study performance and outcomes, while facilitating the research process. Frequently, steps that involve human intervention are the weak links in the process. Risk analysis therefore gives considerable benefit to analytical validation, assessing and avoiding failures due to human error, potential imprecision in applying protocols, uncertainty in equipment function and imperfect control of materials. This paper describes in detail how FMEA methodology can be applied as a performance improvement tool in the field of non-regulated research, specifically on a basic Life Sciences research process. We chose as “pilot process” the selection of oligonucleotide aptamers for therapeutic purposes, as an example of a complex and multi-step process, suitable for technology transfer. We applied FMEA methodology, seeking every opportunity for error and its impact on process output, and then, a set of improvement actions was generated covering most aspects of laboratory practice, such as equipment management and staff training. We also propose a useful tool supporting the risk assessment of research processes and its outputs and that we named “FMEA strip worksheet.” These tools can help scientists working in non-regulated research to approach Quality Management and to perform risk evaluation of key scientific procedures and processes with the final aim to increase and better control efficiency and efficacy of their research.

In recent years there has been growing evidence that all organisms and the environment are exposed to hormone-like chemicals, known as endocrine disruptor chemicals (EDCs). These chemicals may alter the normal balance of endocrine systems and lead to adverse effects, as well as an increasing number of hormonal disorders in the human population or disturbed growth and reduced reproduction in the wildlife species. For some EDCs, there are documented health effects and restrictions on their use. However, for most of them, there is still no scientific evidence in this sense. In order to verify potential endocrine effects of a chemical in the full organism, we need to test it in appropriate model systems, as well as in the fruit fly, Drosophila melanogaster. Here we report detailed in vivo protocols to study endocrine disruption in Drosophila, addressing EDC effects on the fecundity/fertility, developmental timing, and lifespan of the fly. In the last few years, we used these Drosophila life traits to investigate the effects of exposure to 17-α-ethinylestradiol (EE2), bisphenol A (BPA), and bisphenol AF (BPA F). Altogether, these assays covered all Drosophila life stages and made it possible to evaluate endocrine disruption in all hormone-mediated processes. Fecundity/fertility and developmental timing assays were useful to measure the EDC impact on the fly reproductive performance and on developmental stages, respectively. Finally, the lifespan assay involved chronic EDC exposures to adults and measured their survivorship. However, these life traits can also be influenced by several experimental factors that had to be carefully controlled. So, in this work, we suggest a series of procedures we have optimized for the right outcome of these assays. These methods allow scientists to establish endocrine disruption for any EDC or for a mixture of different EDCs in Drosophila, although to identify the endocrine mechanism responsible for the effect, further essays could be needed.

In recent years there has been growing evidence that all organisms and the environment are exposed to hormone-like chemicals, known as endocrine disruptor chemicals (EDCs). These chemicals may alter the normal balance of endocrine systems and lead to adverse effects, as well as an increasing number of hormonal disorders in the human population or disturbed growth and reduced reproduction in the wildlife species. For some EDCs, there are documented health effects and restrictions on their use. However, for most of them, there is still no scientific evidence in this sense. In order to verify potential endocrine effects of a chemical in the full organism, we need to test it in appropriate model systems, as well as in the fruit fly, Drosophila melanogaster. Here we report detailed in vivo protocols to study endocrine disruption in Drosophila, addressing EDC effects on the fecundity/fertility, developmental timing, and lifespan of the fly. In the last few years, we used these Drosophila life traits to investigate the effects of exposure to 17-α-ethinylestradiol (EE2), bisphenol A (BPA), and bisphenol AF (BPA F). Altogether, these assays covered all Drosophila life stages and made it possible to evaluate endocrine disruption in all hormone-mediated processes. Fecundity/fertility and developmental timing assays were useful to measure the EDC impact on the fly reproductive performance and on developmental stages, respectively. Finally, the lifespan assay involved chronic EDC exposures to adults and measured their survivorship. However, these life traits can also be influenced by several experimental factors that had to be carefully controlled. So, in this work, we suggest a series of procedures we have optimized for the right outcome of these assays. These methods allow scientists to establish endocrine disruption for any EDC or for a mixture of different EDCs in Drosophila, although to identify the endocrine mechanism responsible for the effect, further essays could be needed.

Mitochondrial dysfunction seems to play a fundamental role in the pathogenesis of neurodegeneration in Huntington's disease (HD). We assessed possible neuroprotective actions of meldonium, a small molecule affecting mitochondrial fuel metabolism, in in vitro and in vivo HD models. We found that meldonium was able to prevent cytotoxicity induced by serum deprivation, to reduce the accumulation of mutated huntingtin (mHtt) aggregates, and to upregulate the expression of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) in mHTT-expressing cells. The PGC-1α increase was accompanied by the increment of mitochondrial mass and by the rebalancing of mitochondrial dynamics with a promotion of the mitochondrial fusion. Meldonium-induced PGC-1α significantly alleviated motor dysfunction and prolonged the survival of a transgenic HD Drosophila model in which mHtt expression in the nervous system led to progressive motor performance deficits. Our study strongly suggests that PGC-1α, as a master coregulator of mitochondrial biogenesis, energy homeostasis, and antioxidant defense, is a potential therapeutic target in HD.

Background Mitochondrial dysfunction, characterized by abnormalities of mitochondrial biogenesis, dynamics, trafficking, and alterations in the synthesis of high-energy molecules, seems to play a fundamental role in the pathogenesis of neurodegeneration in Huntington’s disease (HD). Meldonium is a small molecule that inhibits endogenous carnitine synthesis and carnitine cell uptake. The decrease of carnitine availability inside the cell induces a significant reprogramming of cell metabolic pathways that apparently counters the mitochondrial dysfunction, at least in the case of heart or brain ischemia. Aims To evaluate neuroprotective actions of meldonium in both in vitro and in vivo HD models. Methods We used STHdhQ111/Q111 cells expressing a mutated form of the huntingtin protein (mHtt), and a transgenic Drosophila model of HD in which the expression of Htt, with a 128Q expansion (Q128HD-FL) in the nervous system, led to progressive motor performance deficits. Results We found that meldonium could prevent cytotoxicity induced by serum deprivation in STHdhQ111/Q111 cells. Meldonium could also reduce the accumulation of mHtt aggregates inside the cell. In addition, meldonium was able to upregulate the expression of PGC-1α, which is a master co-regulator of mitochondrial biogenesis, energy homeostasis, and antioxidant defense, as well as a potential HD target. As expected, the increase of PGC-1α was accompanied by the increment of mitochondrial mass and by the rebalancing of mitochondrial dynamics with a promotion of the mitochondrial fusion. Interestingly, when given per os, meldonium significantly alleviated motor dysfunction and prolonged the survival of transgenic Drosophila model of HD. Conclusions Our study strongly suggests, after considering these factors together, that meldonium (itself or as a lead structure for the synthesis of novel drugs) is a potential treatment for the management of HD.

The scientific world is facing the "quality revolution": not only results have been led to research and social community attention, but also reliability, safety and efficacy of discoveries and efficiency of fund exploitation. Quality management in scientific R&D has become an essential tool in ensuring that modern scientific development is implemented within a rigorous and robust quality framework. The need for Quality standards in non-regulated research is a matter of considerable current debate inside international research community. The qPMO is a research network involving 5 different Institutes and two Departments of the Italian National Research Council (CNR), aimed at realizing a Total Quality Management (TQM) model for Life Sciences laboratories. This TQM OpenLab model will act as a uniform environment in which strong, innovation-oriented research projects can be designed and developed according to international Quality standards and with the planning of Horizon 2020. qPMO covers four different fields to experiment with a specific Quality approach to scientific research and to generate models for the implementation of Quality methodologies in science. Our experience demonstrated that Quality tools can strongly support the management of scientific research through disseminating knowledge, best practice and interoperability and enhance of the economic value of project and research outcomes.

mst36Fa and mst36Fb are two male-specific genes that are part of a novel gene family recently characterized in Drosophila melanogaster. The genes are strictly clustered and show an identical tissue and temporal expression pattern limited to the male germline. Here we demonstrate that the transcription of these two genes, which is triggered by different cis regulatory elements, responds to the same testis-specific factors encoded by the aly and can class meiotic arrest genes. RNA interference was used to decrease expression of these two genes. We obtained a reduction of fertility in the transgenic adult males compared to the wild type. These data suggest that the Mst36Fa and Mst36Fb proteins may have an important role in the production of functional sperm.

The intersex (ix) gene works in concert with doublesex (dsx) at the bottom of the sex-determination hierarchy to control somatic sexual differentiation in Drosophila melanogaster females. Here we report the isolation and characterization of the Drosophila intersex (ix) homologue in the pest lepidopteron Maruca vitrata (Mvix). The Mvix gene exhibits major complexity with respect to the Drosophila homolog. It is expressed in males and females and its pre-mRNA is subject to differential splicing events which affect both the protein coding and the non-coding regions. Moreover, Northern blot experiments revealed the presence of a female-specific transcript in pupae RNA, which appears to be the first described sex specific transcript of ix homologs characterized to date. The expression of Mvix cDNA in D. melanogaster transgenic flies indicates that the MvIX product, which shares a relatively high degree of homology with the D. melanogaster IX protein, is able to partially rescues the Drosophila mutant phenotype.

Design of Experiments (DoE) statistical methodology permits the simultaneous evaluation of the effects of different factors on experimental performance and the analysis of their interactions in order to identify their optimal combinations. Compared to classical approaches based on changing only one factor at a time (OFAT), DoE facilitates the exploration of a broader range of parameters combinations, as well as providing the possibility to select a limited number of combinations covering the whole frame. The advantage of DoE is to maximise the amount of information provided and to save both time and money. DoE has been primarily used in industry to maximise process robustness, but recently it has also been applied in biomedical research to different types of multivariable analyses, from determination of the best cell media composition to the optimisation of entire multi-step laboratory protocols such as cell transfection.

The Design of Experiment (DoE) is a statistical methodology, very useful in the case of multivariable assays, that permits to evaluate simultaneously the influence of different factors on a specific output and to analyse the interactions among them in order to identify their optimal combinations. Moreover, DoE allows to select from the huge numbers of combinations only a limited number, in order to cover the whole frame, allowing to save both time and money. DoE was mostly used in industrial field in order to maximize robust processes, but recently it has been also applied in biomedical research field. Different studies have demonstrated the advantages of using a DoE approach compared to the classical one (a single parameter is tested in each assay) in the context of automated experiments, determination of cell media [1] compositions or HPLC tuning [2]. In the present study, we utilize for the first time the DoE methodology to optimize the transfection protocol of neural cells, as an example of DoE application to a laboratory procedure. Neural precursor cells are hard to transfect and refractory to lipidic reagents [3], for this reason we choose as transfectant reagent the cationic not-lipidic Poliethylenimmine (PEI). The DoE approach allowed us to identify the main variables (factors) affecting the transfection efficiency and to discover their optimal combinations, developing a protocol that let us to triplicate the transfection efficiency respect to the initial conditions. Moreover, the covariance analysis unmasked significant variable interactions impossible to calculate in one factor-variation tests used in normal laboratory practices. Our results indicate that DoE might be very helpful also in research for the identification of the better experimental conditions and the analysis of interactions between different variables.

Quality principles and methodologies have long been ignored in Non Regulated Scientific Research, even though they have been widely used in industrial and business applications in recent decades, improving management and results as well as reducing costs. A groundbreaking project named Quality and Project Management OpenLab (qPMO) was implemented by a cluster of laboratories belonging to the Italian National Research Council (CNR) with the aim of defining efficient models for the application of Quality methodologies to the Life Sciences. For the first time, CNR researchers are working with Quality consultants to apply Quality methodologies to scientific research. Project management and team building techniques (e.g., decision matrix, project charter, debriefing, Plan-Do-Check-Act) were employed to rapidly define a project with four lines of research based on Quality methodologies. Among them, researchers chose to experiment with the definition of standards and guidelines for research activities, experiment optimization via Design of Experiment (DoE), management of a research lab via an ISO9001 Quality Management System, support to technology transfer via Failure Mode and Effect Analysis (FMEA). All of these aspects converge in a model of Total Quality Management OpenLab which is disseminated through a web platform specifically developed for this project. Further, the acquired habits of acting in organized teams, using group management tools and information technologies, supported the work of each group in their respective research line. The results show better use of time and project consistency: the project was set up in one month, and the four research lines accomplished their planned milestones in advance with respect to the initial timetable. Emphasis was also made on Quality training that created a common management and control culture and brought to researchers' attention tools and methodologies widely used in other fields. Each line of activity also brought innovative and promising Quality approaches to scientific research.

Quality principles and methodologies can strongly support the management of scientific research, in both basic and applied research laboratories, where procedures and results are rapidly changing and can hardly be standardized. In this view, Quality methodologies, such as Failure Mode and Effect Analysis - FMEA, was borrowed from the industrial field, where it is widely used in risk control and process optimization procedures, to validate and support research activities and results, to create a standard and controlled workplace, and to support the interaction between research and industrial application.We applied FMEA analysis on a "pilot" process, developed for the selection of cell-specific aptamers, in agreement with the needs of companies interested in the development of this methodology.

The Pre-intermoult gene-1 (Pig-1) of Drosophila melanogaster maps on the X chromosome, at polytene bands 3C11-12, and is nested within the 79 kb intron of the dunce gene. Pig-1 has so far been characterized only preliminarily and its function is still unknown. We analysed the molecular organization of the gene by cDNA clone isolation and sequencing as well as S1 mapping and primer extension analyses. The results obtained reveal that the gene is colinear with its genomic sequence and define the usage of both 5' and 3' alternative sites for Pig-1 transcription; two continuous open reading frames (ORFs) are fully contained within the Pig-1 transcribed region, although several lines of evidence suggest that only the longer ORF is likely to be translated. We also report that the level of Pig-1 transcript is nearly fourfold reduced in a variant strain carrying a deletion within the Pig-1 upstream sequence, thus identifying a regulatory element required for high level gene expression.

The molecular organization of the dunce gene of Drosophila melanogaster has proved to be particularly complex, with two divergently transcribed genes, Sgs-4 and Pig-1, nested within its 79 kb intron (1). Here we report the identification and the molecular characterization of a third gene nested within the transcription unit of dunce. This newly identified gene is located nearly 6 kb downstream Pig-1, within a more upstream dunce intron. The gene is developmentally regulated and transcribed with the same polarity of dunce; several lines of evidence indicate that it might encode for a salivary gland secreted (Sgs) protein.

The X-linked Sgs-4 gene of Drosophila melanogaster encodes a salivary glue protein. Here we report the molecular characterization of a non-dosage compensated variant strain, named Karsnas, in which males accumulate only about half of the Sgs-4 polypeptide amount as do females. The results obtained show that significant nucleotide sequence alterations are accumulated within the Sgs-4 coding and 3' untranslated region of the variant strain, thus suggesting a possible role of these sequences in the Sgs-4 dosage compensation.

SummaryThe Drosophila melanogasterecd1 mutation causes a severe temperature-sensitive deficiency in the titre of the steroid hormone eedysone. This mutation was used to investigate the role of eedysone in both the transcription of the genes mapped at the 3C11–12 intermoult puff region and the puff formation. Thoroughly synchronized ecd1 larvae were shifted to the non-permissive temperature at various times of the development; after 24 or 48 h, the levels of the transcripts derived from Sgs-4, Pig-1 and ng-1, the three genes located at the 3C11–12 polytene bands, were determined. The results showed that the levels of the transcripts encoded by Pig-1 and ng-1 are unaffected by the drop in the ecdysone titre occurring in non-permissive conditions whereas the amount of Sgs-4 mRNA is greatly reduced. These data clearly indicate that transcription of the three genes mapped within the puff region is affected differently by the hormone. Furthermore, ecd1 larvae cultured at the non-permissive temperature show a prominent puff at the 3C11–12 polytene bands, indicating that eedysone is not essential for puff induction and that puff size is not simply correlated with high-level Sgs-4 transcription.

We describe here a Drosophila gene, tosca (tos), that is specifically expressed in the female germline. tos mRNA accumulates selectively within the pro-oocyte in germarial region 2 and persists throughout oogenesis. In the early embryo, the maternally supplied tos mRNA is evenly distributed at the syncytial blastoderm stage, but is excluded from the forming cells when cellularization begins. tos product is the first Drosophila member of the RAD2 protein family, a group of related DNA repair nucleases conserved from yeast to humans. Within the family, Tos is more closely related to ExoI, a Schizosaccharomyces pombe 5'--3' double-stranded DNA exonuclease specifically induced in meiotic prophase I. The definite oocyte localization of tos transcript during meiosis and its ubiquitous distribution in early embryos suggest that tos may play a role in mismatch repair during genetic recombination and early cleavage divisions.