Combined flow cytometry and high-throughput image analysis for the study of essential genes in Caenorhabditis elegans

Background: Advances in automated image-based microscopy platforms coupled with high-throughput liquid workflows have facilitated the design of large-scale screens utilising multicellular model organisms such as Caenorhabditis elegans to identify genetic interactions, therapeutic drugs or disease modifiers. However, the analysis of essential genes has lagged behind because lethal or sterile mutations pose a bottleneck for high-throughput approaches, and a systematic way to analyse genetic interactions of essential genes in multicellular organisms has been lacking. Results: In C. elegans, non-conditional lethal mutations can be maintained in heterozygosity using chromosome balancers, commonly expressing green fluorescent protein (GFP) in the pharynx. However, gene expression or function is typically monitored by the use of fluorescent reporters marked with the same fluorophore, presenting a challenge to sort worm populations of interest, particularly at early larval stages. Here, we develop a sorting strategy capable of selecting homozygous mutants carrying a GFP stress reporter from GFP-balanced animals at the second larval stage. Because sorting is not completely error-free, we develop an automated high-throughput image analysis protocol that identifies and discards animals carrying the chromosome balancer. We demonstrate the experimental usefulness of combining sorting of homozygous lethal mutants and automated image analysis in a functional genomic RNA interference (RNAi) screen for genes that genetically interact with mitochondrial prohibitin (PHB). Lack of PHB results in embryonic lethality, while homozygous PHB deletion mutants develop into sterile adults due to maternal contribution and strongly induce the mitochondrial unfolded protein response (UPR mt ). In a chromosome-wide RNAi screen for C. elegans genes having human orthologues, we uncover both known and new PHB genetic interactors affecting the UPR mt and growth. Conclusions: The method presented here allows the study of balanced lethal mutations in a high-throughput manner. It can be easily adapted depending on the user's requirements and should serve as a useful resource for the C. elegans community for probing new biological aspects of essential nematode genes as well as the generation of more comprehensive genetic networks.European Research Council ERC-2011-StG-281691Ministerio de Economía y Competitividad BFU2012–3550

Vulnerabilities and the Italian Protection System: An ethnographic exploration of the perspectives of protection seekers

The following research is part of the Horizon2020 project “Vulnerabilities under the Global Protection Regime. How does the law assess, address, shape, and produce the vulnerabilities of protection seekers?” (VULNER, GA 870845) and focuses on the Italian context. The first report (Vulnerabilities in the asylum and protection system in Italy), based on the research conducted in 2020, focused on the documentation and analysis of the existing legal and bureau- cratic mechanisms in place, to identify and assess the vulnerabilities among “protection seek- ers”, meaning all migrants seeking protection, regardless of the legal status they have eventually achieved.1 In this first report, we compared the ways in which Italian legislation and case-law approach vulnerabili- ties and how it arranges legal and bureaucratic instruments for recognition and protection, with the di- rect experience of legal experts and of those who, at different levels, make decisions on which situations of vulnerability can find effective legal recognition. On the one hand, our qualitative research deepened our understanding of the vulnerability assessment processes, as well as of the perspective of those who intervene in situations of vulnerability (decision makers, international organizations, legal actors, etc). On the other, the report highlights the effects of the transposition of these measures into concrete practices, both in terms of the most virtuous applications implemented in various territorial contexts and the dis- connect between “law on paper” and “law in practice.” In this second report, based on ethnographic research conducted in 2021, the focus shifts to the direct experiences of protection seekers and of those who work in close contact with them. Throughout the research, we investigated what opinion protection seekers as well as people working in reception and support services had about the protection system in Italy, both in terms of procedures and identification, protection, reception, and support measures provided for people in vulnerable sit- uations. The inclusion of an operational and “bottom-up” point of view made it possible to check, vali- date and integrate what emerged in the previous report. The fieldwork took place between April 2021 and February 2022. We conducted 64 in-depth interviews, ethnographic observations in several reception and support centers for migrant people, as well as multi- ple informal conversations with at least 200 people - including protection seekers and social workers. The work was carried out in two Italian regions (Veneto and Lazio) and it shed light on series of issues that cut across the two contexts – and thus are of national relevance – as much as on issues that were distinct to the local context. The research shows how it is essential to consider the complex issue of vulnerabilities related to migration experiences through qualitative research methods that go beyond quantitative data. The ethnographic approach chosen within the framework of this research made evident the importance of investigating the issue of vulnerabilities through a methodology capable of bringing out the heterogeneity of situa- tions, the social transformations taking place and the intertwining of several factors (personal, social, ge- 1 In line with the conceptual framework that guides the VULNER project, the category ‘protection seekers’ is conceived to also include migrants seeking protection but who do not necessarily fit the definition and requirements for applying for international protection. In the Italian context, we focus on migrants who are asylum seekers or having alternative types of protection (for instance related to human trafficking, gender-based violence, age, social inclusion, etc.), as well as on actual undocumented migrants whose applications on these grounds have been rejected. 4 Carnassale, D., Marchetti, S., 2022 ographic, situational). The qualitative approach shed light, from the perspective of those involved, on the impact of broader issues – such as the functioning of the protection system at the European and national level or the functioning of local services and administrations – on individual experiences. The research brings to light how situations of vulnerability may be understood differently by peo- ple seeking protection than by social workers and other legal and institutional actors, as well as how these situations may be pre-existing or become evident only at a specific moment in the mi- gration journey. The report devotes particular attention to how multiple situations of vulnerability are deeply connected to subjective conditions, migration trajectories, living conditions, and protection seekers’ agency. The research also shows how various situations of vulnerability can take on different meanings and forms depending on whether they are related to the country of origin, to the journey, to the arrival in Italy, to a long stay in the destination context, or to additional problems caused by having moved to other Euro- pean countries. One section of the report investigates how the reforms introduced in Italy between 2017 and 2020 on immigration law addressed protection seekers’ experiences of vulnerability, but also contrib- uted in fostering them. Ethnography made it possible to highlight the daily life experiences of people who live or have lived in highly precarious situations, or who experienced forms of control or institutional abandonment. The report highlights a number of critical issues that need to be addressed in the future. Indeed, the research results made evident how changes in regulations and their effects on procedures and re- ception services have had a profound impact on territories, services and protection seekers themselves. The research documented how regulatory actions have often had a vulnerabilizing effect on protection seekers, while they do not seem to have facilitated the identification, recognition and protection of many situations of vulnerability described by migrants interviewed. The picture that emerges from our work is that several vulnerabilities deemed particularly serious find formal but not necessarily concrete and material recognition, while others tend to be ignored or at least underestimated. This has a negative spillover effect both in the lives of migrant people and on the work of local services and administrations. Furthermore, we found out that, in recent years, several critical issues that already existed in the system worsened, while the inability to intervene adequately in favor of full recognition and protection of situations of fragility increased. In our view, it is therefore necessary to intervene at the institutional level and enhance the point of view of protection seekers and social workers, including those working in the non-profit sector

CRF-like receptor SEB-3 in sex-common interneurons potentiates stress handling and reproductive drive in C. elegans

Environmental conditions can modulate innate behaviours. Although male Caenorhabditis elegans copulation can be perturbed in the presence of stress, the mechanisms underlying its decision to sustain copulation are unclear. Here we describe a mating interference assay, which quantifies the persistence of male C. elegans copulation in noxious blue light. We show that between copulations, the male escapes from blue light illumination at intensities over 370 μW mm(−2). This response is attenuated in mutants with constitutive activation of the corticotropin-releasing factor receptor family homologue SEB-3. We show that activation of this receptor causes sex-common glutamatergic lumbar ganglion interneurons (LUA) to potentiate downstream male-specific reproduction circuits, allowing copulatory behaviours to partially override the light-induced escape responses in the male. SEB-3 activation in LUA also potentiates copulation during mild starvation. We suggest that SEB-3 activation allows C. elegans to acclimate to the environment and thus continue to execute innate behaviours even under non-optimal conditions

September 27, 1994 University Chronicle

Shawnee State University Student Newspaperhttps://digitalcommons.shawnee.edu/chronicle/1072/thumbnail.jp

Traffic and Related Self-Driven Many-Particle Systems

Since the subject of traffic dynamics has captured the interest of physicists, many astonishing effects have been revealed and explained. Some of the questions now understood are the following: Why are vehicles sometimes stopped by so-called ``phantom traffic jams'', although they all like to drive fast? What are the mechanisms behind stop-and-go traffic? Why are there several different kinds of congestion, and how are they related? Why do most traffic jams occur considerably before the road capacity is reached? Can a temporary reduction of the traffic volume cause a lasting traffic jam? Under which conditions can speed limits speed up traffic? Why do pedestrians moving in opposite directions normally organize in lanes, while similar systems are ``freezing by heating''? Why do self-organizing systems tend to reach an optimal state? Why do panicking pedestrians produce dangerous deadlocks? All these questions have been answered by applying and extending methods from statistical physics and non-linear dynamics to self-driven many-particle systems. This review article on traffic introduces (i) empirically data, facts, and observations, (ii) the main approaches to pedestrian, highway, and city traffic, (iii) microscopic (particle-based), mesoscopic (gas-kinetic), and macroscopic (fluid-dynamic) models. Attention is also paid to the formulation of a micro-macro link, to aspects of universality, and to other unifying concepts like a general modelling framework for self-driven many-particle systems, including spin systems. Subjects such as the optimization of traffic flows and relations to biological or socio-economic systems such as bacterial colonies, flocks of birds, panics, and stock market dynamics are discussed as well.Comment: A shortened version of this article will appear in Reviews of Modern Physics, an extended one as a book. The 63 figures were omitted because of storage capacity. For related work see http://www.helbing.org

Home between bidesh and shodesh: Domestication of Living Spaces, Identity and Gender Experiences in the Bangladeshi Diaspora

This article deals with the housing strategies and changing living styles of the Bangladeshi population in a small town in north-eastern Italy. It analyses the re-use and “domestication” of everyday public spaces, as a way of exploring how bidesh (foreign-land) space is transformed into a shodesh, home-like space. A parallel process of re-functionalization occurs in the private sphere. Different forms of cohabitation are put in place to deal with immigrants’ family-based needs, against deteriorating economic conditions. The process is not without contradictions. For instance, family reunification allows men to recover an important component of their emotional universe, possibly healing the loneliness of migration. Reunified women, though, may experience their new home as an ambivalent place of solitude. Along these lines, the paper also highlights the gap between men’s and women’s views of “home” and “homeland”, in order to make sense of their evolving ways of “feeling at home”