Reverse genetic strategies in Caenorhabditis elegans: towards controlled manipulation of the genome.

Caenorhabditis elegans has a complete annotated genome sequence that is augmented by increasing quantities of data from high-throughput postgenomic analyses. This has led to an increasing need to identify the biological functions of specific genes using reverse genetics, i.e., moving from gene to phenotype. Fundamental to this aim is the ability to alter the structure of particular genes by means that are not accessible to classical genetic strategies. Thus, one dream of C. elegans researchers is to establish a toolkit for the controlled manipulation of any loci within the genome. Although C. elegans is amenable to a wide variety of genetic and molecular manipulations, controlled manipulation of endogenous genes by, for example, gene targeting has proved elusive until relatively recently. In this review, we describe and discuss the different methods available for the inactivation and modification of endogenous loci with a focus on strategies that permit some measure of control in this process. We describe methods that use random mutagenesis to isolate mutations in specific genes. We then focus on techniques that allow controlled manipulation of the genome: gene modification by transposon mobilisation, gene knock-out mediated by zinc-finger nucleases, and gene targeting by biolistic transformation

Multiview motion tracking based on a cartesian robot to monitor Caenorhabditis elegans in standard Petri dishes

[EN] Data from manual healthspan assays of the nematode Caenorhabditis elegans (C. elegans) can be complex to quantify. The first attempts to quantify motor performance were done manually, using the so-called thrashing or body bends assay. Some laboratories have automated these approaches using methods that help substantially to quantify these characteristic movements in small well plates. Even so, it is sometimes difficult to find differences in motor behaviour between strains, and/or between treated vs untreated worms. For this reason, we present here a new automated method that increases the resolution flexibility, in order to capture more movement details in large standard Petri dishes, in such way that those movements are less restricted. This method is based on a Cartesian robot, which enables high-resolution images capture in standard Petri dishes. Several cameras mounted strategically on the robot and working with different fields of view, capture the required C. elegans visual information. We have performed a locomotion-based healthspan experiment with several mutant strains, and we have been able to detect statistically significant differences between two strains that show very similar movement patterns.This work was supported by the research agency of the Spanish Ministry of Science and Innovation under Grant RTI2018-094312-B-I00 (European FEDER funds).Puchalt-Rodríguez, JC.; González-Rojo, JF.; Gómez-Escribano, AP.; Vázquez-Manrique, RP.; Sánchez Salmerón, AJ. (2022). Multiview motion tracking based on a cartesian robot to monitor Caenorhabditis elegans in standard Petri dishes. Scientific Reports. 12(1):1-11. https://doi.org/10.1038/s41598-022-05823-611112

IP3 signalling regulates exogenous RNAi in Caenorhabditis elegans.

RNA interference (RNAi) is a widespread and widely exploited phenomenon. Here, we show that changing inositol 1,4,5-trisphosphate (IP3) signalling alters RNAi sensitivity in Caenorhabditis elegans. Reducing IP3 signalling enhances sensitivity to RNAi in a broad range of genes and tissues. Conversely up-regulating IP3 signalling decreases sensitivity. Tissue-specific rescue experiments suggest IP3 functions in the intestine. We also exploit IP3 signalling mutants to further enhance the sensitivity of RNAi hypersensitive strains. These results demonstrate that conserved cell signalling pathways can modify RNAi responses, implying that RNAi responses may be influenced by an animal's physiology or environment.We thank A. Fire, K. Ford, S. Mitani and H. Peterkin for the provision of plasmids and strains. Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). Other strains were provided by the Mitani Lab through the National Bio‐Resource Project of the MEXT, Japan. We are grateful to J. Ahringer, B. Olofsson and members of the Baylis group for helpful discussions. AIN was funded by Trinity Hall College, Cambridge and the Cambridge European Trust. The work of MDS and RPV‐M was partially funded by a Miguel Servet Grant (CP11/00090) from the Health Research Institute Carlos III, which is partially supported by the European Regional Development Fund. RPV‐M is a Marie Curie fellow (CIG322034). RG was funded by the MRC (G0601106).This is the accepted manuscript. The final version is available from Embo Press at http://embor.embopress.org/content/early/2015/01/21/embr.201439585

Synergistic activation of AMPK prevents from polyglutamine-inducedtoxicity inCaenorhabditis elegans

11 páginas, 4 figuras. Supplementary material related to this article can be found, in the online version, at doi: https://doi.org/10.1016/j.phrs.2020.105105.Expression of abnormally long polyglutamine (polyQ) tracks is the source of a range of dominant neurodegenerative diseases, such as Huntington disease. Currently, there is no treatment for this devastating disease, although some chemicals, e.g., metformin, have been proposed as therapeutic solutions. In this work, we show that metformin, together with salicylate, can synergistically reduce the number of aggregates produced after polyQ expression in Caenorhabditis elegans. Moreover, we demonstrate that incubation polyQ-stressed worms with low doses of both chemicals restores neuronal functionality. Both substances are pleitotropic and may activate a range of different targets. However, we demonstrate in this report that the beneficial effect induced by the combination of these drugs depends entirely on the catalytic action of AMPK, since loss of function mutants of aak-2/AMPKα2 do not respond to the treatment. To further investigate the mechanism of the synergetic activity of metformin/salicylate, we used CRISPR to generate mutant alleles of the scaffolding subunit of AMPK, aakb-1/AMPKβ1. In addition, we used an RNAi strategy to silence the expression of the second AMPKβ subunit in worms, namely aakb-2/AMPKβ2. In this work, we demonstrated that both regulatory subunits of AMPK are modulators of protein homeostasis. Interestingly, only aakb-2/AMPKβ2 is required for the synergistic action of metformin/salicylate to reduce polyQ aggregation. Finally, we showed that autophagy acts downstream of metformin/salicylate-related AMPK activation to promote healthy protein homeostasis in worms.We thank the CGC, funded by the NIH Office of ResearchInfrastructure Programs (P40 OD010440), for worm strains. [...] RPVMis aMiguel Servet type IIresearcher (CPII16/00004) funded by Institutode Salud Carlos III (ISCIII, Madrid, Spain). Grants from the ISCIII wereused to perform this work (PI14/00949 and PI17/00011). All grantsfrom ISCIII are co-financed by the European Development RegionalFund”A way to achieve Europe”(ERDF). JBY holds a grant from theGeneralitat Valenciana and the European Social Fund (ACIF/2019/249). Some equipment used in this work has been funded in partnershipbetween the Generalitat Valenciana (Conselleria de Sanitat I SalutPública, Valencian Community, Spain) and European Funds (ERDF/FSE), through the call "Improvement of research infrastructures for rarediseases”CV FEDER 2014-2020. This work has been partially supportedby a grant from the Fundació Telemarató de la TV3 (Reference 559),which covered the work of MDS. The funds from the ISCIII are partiallysupported by the European Regional Development Fund. RPVM is also aMarie Curie fellow (CIG322034, EU). This work has been partiallysupported by a grant from the CIBERER (ACCI2016), a grant from theFundación Ramón Areces (CIVP19S8119) and anAyuda Miguel Gilgrantto RPVM (VII Convocatoria Ayudas a la Investigación MHER, 2019Peer reviewe

Role of glutathione reductase in proteostasis regulation

Trabajo presentado en la V Spanish Worm Meeting, celebrada en Salamanca el 5 y 6 de marzo de 2015.Peer Reviewe

An Integrated Intervention Model for the Prevention of Zika and Other Aedes-Borne Diseases in Women and their Families in Mexico

We describe and discuss the rationale, design and current implementation of a model for an integrated intervention for the primary and secondary prevention of Zika and other Aedes-borne diseases and sexually transmitted infections that impact reproductive health, pregnancy and perinatal life stages in women and their families in Merida, Mexico. The intervention includes enhanced surveillance of pregnant women, implementation of communication strategies to promote good practices to prevent disease transmission, determination of the frequency of structural anomalies detected prenatally in the foetus, umbilical cord and placenta of pregnancies diagnosed with ZIK infection, detection of ZIKV and other arboviruses/viruses in products of abortion, in-utero and early newborn, provision of Aedes aegypti-proof houses? (protecting homes with door/window screens with insecticide to prevent the access of mosquitoes), mosquito repellents, larvicides and education/promotion of best practices for the prevention of infection with dengue (DENV), chikungunya (CHIKV) and Zika (ZIKV) and an anthropological studies on sociocultural factors of pregnant women associated with ZIKV. This intervention is being developed in a population of 10,000 people of the city of Merida and with the participation of a multidisciplinary group of public health professionals in collaboration with the Ministry of Health and the Government of Yucatan

Loss of glutathione redox homeostasis impairs proteostasis by inhibiting autophagy-dependent protein degradation

In the presence of aggregation-prone proteins, the cytosol and endoplasmic reticulum (ER) undergo a dramatic shift in their respective redox status, with the cytosol becoming more oxidized and the ER more reducing. However, whether and how changes in the cellular redox status may affect protein aggregation is unknown. Here, we show that C. elegans loss-of-function mutants for the glutathione reductase gsr-1 gene enhance the deleterious phenotypes of heterologous human, as well as endogenous worm aggregation-prone proteins. These effects are phenocopied by the GSH-depleting agent diethyl maleate. Additionally, gsr-1 mutants abolish the nuclear translocation of HLH-30/TFEB transcription factor, a key inducer of autophagy, and strongly impair the degradation of the autophagy substrate p62/SQST-1::GFP, revealing glutathione reductase may have a role in the clearance of protein aggregates by autophagy. Blocking autophagy in gsr-1 worms expressing aggregation-prone proteins results in strong synthetic developmental phenotypes and lethality, supporting the physiological importance of glutathione reductase in the regulation of misfolded protein clearance. Furthermore, impairing redox homeostasis in both yeast and mammalian cells induces toxicity phenotypes associated with protein aggregation. Together, our data reveal that glutathione redox homeostasis may be central to proteostasis maintenance through autophagy regulation.. The Spanish Ministry of Economy and Competitiveness supported EF-S and VG (BFU2016–78265-P), PA (BFU2016– 79313-P and MDM-2016–0687), and AM-V (BFU2015–64408-P). AM-V was also supported by the Instituto de Salud Carlos III (PI11/ 00072) and RPV-M (CPII16/00004, PI14/00949 and PI17/00011). All projects were cofinanced by the Fondo Social Europeo (FEDER). AM-V is a member of the GENIE and EU-ROS Cost Actions of the European Union and RPV-M is a Marie Curie Fellow (CIG322034, EU)

Recommendations for screening, monitoring, prevention, and prophylaxis of infections in adult and pediatric patients receiving CAR T-cell therapy : a position paper

Chimeric antigen receptor (CAR) T-cell therapy is one of the most promising emerging treatments for B-cell malignancies. Recently, two CAR T-cell products (axicabtagene ciloleucel and tisagenlecleucel) have been approved for patients with aggressive B-cell lymphoma and acute lymphoblastic leukemia; many other CAR-T constructs are in research for both hematological and non-hematological diseases. Most of the patients receiving CAR-T therapy will develop fever at some point after infusion, mainly due to cytokine release syndrome (CRS). The onset of CRS is often indistinguishable from an infection, which makes management of these patients challenging. In addition to the lymphodepleting chemotherapy and CAR T cells, the treatment of complications with corticosteroids and/or tocilizumab increases the risk of infection in these patients. Data regarding incidence, risk factors and prevention of infections in patients receiving CAR-T cell therapy are scarce. To assist in patient care, a multidisciplinary team from hospitals designated by the Spanish Ministry of Health to perform CAR-T therapy prepared these recommendations. We reviewed the literature on the incidence, risk factors, and management of infections in adult and pediatric patients receiving CAR-T cell treatment. Recommendations cover different areas: monitoring and treatment of hypogammaglobulinemia, prevention, prophylaxis, and management of bacterial, viral, and fungal infections as well as vaccination prior and after CAR-T cell therapy

Inositol 1,4,5-Trisphosphate Signalling Regulates the Avoidance Response to Nose Touch in Caenorhabditis elegans

When Caenorhabditis elegans encounters an unfavourable stimulus at its anterior, it responds by initiating an avoidance response, namely reversal of locomotion. The amphid neurons, ASHL and ASHR, are polymodal in function, with roles in the avoidance responses to high osmolarity, nose touch, and both volatile and non-volatile repellents. The mechanisms that underlie the ability of the ASH neurons to respond to such a wide range of stimuli are still unclear. We demonstrate that the inositol 1,4,5-trisphosphate receptor (IP3R), encoded by itr-1, functions in the reversal responses to nose touch and benzaldehyde, but not in other known ASH-mediated responses. We show that phospholipase Cβ (EGL-8) and phospholipase Cγ (PLC-3), which catalyse the production of IP3, both function upstream of ITR-1 in the response to nose touch. We use neuron-specific gene rescue and neuron-specific disruption of protein function to show that the site of ITR-1 function is the ASH neurons. By rescuing plc-3 and egl-8 in a neuron-specific manner, we show that both are acting in ASH. Imaging of nose touch–induced Ca2+ transients in ASH confirms these conclusions. In contrast, the response to benzaldehyde is independent of PLC function. Thus, we have identified distinct roles for the IP3R in two specific responses mediated by ASH