Towards a Mathematical Model of Motility Using \u3ci\u3eDictyostelium discoideum\u3c/i\u3e: Proteins and Geometric Features that Regulate Bleb-Based Motility

A variety of biological functions depend on actin organization. The organization of actin is tightly regulated by a plethora of extracellular and intracellular signaling, scaffolding, and actin-binding proteins. Dysfunctions in this regulation lead to immune diseases, increased susceptibility to pathogens, neurodegenerative diseases, developmental disorders, and cancer metastasis. A variety of actin-dependent processes, including cell motility, are regulated by several proteins of interest: Paxillin, a scaffolding protein; WASP, an actin nucleating protein; SCAR/WAVE, another WASP family actin nucleating protein; Talin, a cortex-to-membrane binding protein; Myosin II, an F-actin contracting motor protein; and Protein Kinase C, a protein kinase. D. discoideum cells have orthologues of all these proteins: PaxB, WasA, SCAR, TalA, MhcA, and PkcA, respectively. Like in mammalian cells, PaxB, WasA, and SCAR all regulate similar actin-based processes. These proteins may regulate each other to execute these actin-based processes. Here, we evaluated WasA and SCAR’s epistatic relationship with PaxB in several actin-based processes using classical, genetic epistasis analysis, and further investigated PaxB, WasA, SCAR, TalA, MhcA, and PkcA’s roles in regulating three dimensional motility where cells endure compression from the environment from other cells and tissue. In three dimensional environments, cells use blebs where the membrane of the cell detaches from the cortex, resulting in a fluid filled blister-like protrusion that dominates motility instead of using filopodia, pseudopodia, and lamellipodia, actin-based structures, that predominate two dimensional motility where cells crawl freely on slides. Most of what we know about these proteins were from studies on slides. Little is known about proteins that regulate blebs and the transition from using actin-based motility structures for blebs. For epistasis, we found that WasA is downstream of PaxB for plaque formation, endocytosis, cell-substrate adhesion, and for cell autonomy. However, mixing experiments revealed that WasA null cells developmental defect could not be rescued by the addition of wild-type cells. We found that SCAR is downstream of PaxB for plaque formation, endocytosis, development, and cell-sorting throughout development. For development specifically, we found that SCAR is a suppressor of PaxB’s null phenotype. In contrast to WasA, SCAR and PaxB separately regulate adhesion. For three dimensional motility and blebs, we used an under agarose assay where null lines of all the actin regulators of interest expressing LifeAct-GFP chemotaxed towards cAMP while being compressed by a RITC-dextran laced gel to induce and visualize blebs. We compared motility under two different agarose concentrations. We found that WT cells increase their use of blebs at the expense of actin-based motility structures from low pressure to high pressure. We found that PaxB negatively regulates blebs and the total number of structures a cell can generate. WasA null cells do not respond to cAMP and would not go under the gel. SCAR positively regulates blebs and negatively regulates pseudopodia under lower pressures. TalA is needed to allow the cells to bleb efficiently under any pressure. MhcA is needed for blebs to form and negatively regulates pseudopodia and lamellipodia. PkcA negatively regulates pseudopodia and lamellipodia under either pressure. Lastly, through our software, we found that negative curvature was a predictor of bleb site location only fifty percent of the time. This investigation has allowed us to advance the fields of actin regulation, signal transduction, cytoskeletal reorganization, cell motility, and their contributions to disease

A role for myosin II clusters and membrane energy in cortex rupture for Dictyostelium discoideum

Blebs, pressure driven protrusions of the cell membrane, facilitate the movement of eukaryotic cells such as the soil amoeba Dictyostelium discoideum , white blood cells and cancer cells. Blebs initiate when the cell membrane separates from the underlying cortex. A local rupture of the cortex, has been suggested as a mechanism by which blebs are initiated. However, much clarity is still needed about how cells inherently regulate rupture of the cor- tex in locations where blebs are expected to form. In this work, we examine the role of mem- brane energy and the motor protein myosin II (myosin) in facilitating the cell driven rupture of the cortex. We perform under-aga rose chemotaxis experiments, using Dictyostelium discoideum cells, to visualize the dynamics of myosin and calculate changes in membrane energy in the blebbing region. To facilitate a rapid detection of blebs and analysis of the energy and myosin distribution at the cell front, we introduce an autonomous bleb detection algorithm that takes in discrete cell boundaries and returns the coordinate location of blebs with its shape characteristics. We are able to identify by microscopy naturally occurring gaps in the cortex prior to membrane detachment at sites of bleb nucleation. These gaps form at posi- tions calculated to have high membrane energy, and are associated with areas of myosin enrichment. Myosin is also shown to accumulate in the cortex prior to bleb initiation and just before the complete disassembly of the cortex. Together our findings provide direct spatial and temporal evidence to support cortex rupture as an intrinsic bleb initiation mechanism and suggests that myosin clusters are associated with regions of high membrane energy where its contractile activity leads to a rupture of the cortex at points of maximal energy

La imagen y la narrativa como herramientas para el abordaje psicosocial en escenarios de violencia. Municipios de Boyacá

(Imágenes)En el presente informe, se evidencian las afectaciones psicosociales en el tejido y la cohesión social en Colombian tras la instauración de los partidos políticos y la incidencia del conflicto armado en el territorio, se busca reconocer los impactos que han dejado los eventos de violencia, los daños, huellas y agravios a la integridad y bienestar de los colectivos por este fenómeno social. Tras la necesidad de generar estrategias para la restitución de derechos hacia quienes han sido víctimas del conflicto armado en el país, inicialmente se analizará el caso de Shimaia mostrando su experiencia como víctima de desplazamiento forzado desde la narrativa; se reconoce la construcción subjetiva de memorias, la incidencia de la experiencia y las afectaciones a la integridad de personas víctimas por este flojeo; para esto, se plantea una serie de preguntas reflexivas, circulares y estrategias en ánimo de propiciar acciones para posibilitar que dichas víctimas trasciendan tal adversidad. Posteriormente se realiza el análisis psicosocial del caso la masacre del Salado en el departamento de Bolívar, se destacan las subjetividades, la construcción y resignificación de memorias, y así mismo los emergentes psicosociales presentes, con el fin de generar estrategias de acompañamiento psicosocial para la potenciación de recursos de afrontamiento en ánimo de mitigar las afectaciones bio-psico-sociales a nivel colectivo e individual. Por último, se desarrolla un ejercicio de foto voz dando a conocer las realidades sociales sobre las violencias identificadas en las comunidades del departamento de Boyacá, especialmente de los municipios de Sogamoso, Tasco, Togüi y Tunja.In this report, the psychosocial effects on the fabric and social cohesion in Colombian after the establishment of political parties and the incidence of the armed conflict in the territory are evidenced, it seeks to recognize the impacts left by events of violence, the damage, traces and grievances to the integrity and well-being of the groups due to this social phenomenon. After the need to generate strategies for the restitution of rights to those who have been victims of the armed conflict in the country, the case of Shimaia will be analyzed initially, showing her experience as a victim of forced displacement from the narrative; the subjective construction of memories, the incidence of experience and the affectations to the integrity of victims of this laziness are recognized; For this, a series of reflective questions, circulars and strategies are proposed in the spirit of promoting actions to enable said victims to transcend such adversity. Subsequently, the psychosocial analysis of the case of the Salado massacre in the department of Bolívar is carried out, highlighting the subjectivities, the construction and redefinition of memories, and likewise the present psychosocial emergents, in order to generate psychosocial accompaniment strategies for empowerment. of coping resources in the spirit of mitigating the bio-psycho-social affectations at the collective and individual level. Finally, a photovoice exercise is developed, making known the social realities about the violence identified in the communities of the department of Boyacá, especially in the municipalities of Sogamoso, Tasco, Togüi and Tunja

Eligibility criteria for Menopausal Hormone Therapy (MHT): a position statement from a consortium of scientific societies for the use of MHT in women with medical conditions. MHT Eligibility Criteria Group

This project aims to develop eligibility criteria for menopausal hormone therapy (MHT). The tool should be similar to those already established for contraception A consortium of scientific societies coordinated by the Spanish Menopause Society met to formulate recommendations for the use of MHT by women with medical conditions based on the best available evidence. The project was developed in two phases. As a first step, we conducted 14 systematic reviews and 32 metanalyses on the safety of MHT (in nine areas: age, time of menopause onset, treatment duration, women with thrombotic risk, women with a personal history of cardiovascular disease, women with metabolic syndrome, women with gastrointestinal diseases, survivors of breast cancer or of other cancers, and women who smoke) and on the most relevant pharmacological interactions with MHT. These systematic reviews and metanalyses helped inform a structured process in which a panel of experts defined the eligibility criteria according to a specific framework, which facilitated the discussion and development process. To unify the proposal, the following eligibility criteria have been defined in accordance with the WHO international nomenclature for the different alternatives for MHT (category 1, no restriction on the use of MHT; category 2, the benefits outweigh the risks; category 3, the risks generally outweigh the benefits; category 4, MHT should not be used). Quality was classified as high, moderate, low or very low, based on several factors (including risk of bias, inaccuracy, inconsistency, lack of directionality and publication bias). When no direct evidence was identified, but plausibility, clinical experience or indirect evidence were available, "Expert opinion" was categorized. For the first time, a set of eligibility criteria, based on clinical evidence and developed according to the most rigorous methodological tools, has been defined. This will provide health professionals with a powerful decision-making tool that can be used to manage menopausal symptoms

HIV-1 drug resistance before initiation or re-initiation of first-line antiretroviral therapy in low-income and middle-income countries: a systematic review and meta-regression analysis.

BACKGROUND: Pretreatment drug resistance in people initiating or re-initiating antiretroviral therapy (ART) containing non-nucleoside reverse transcriptase inhibitors (NNRTIs) might compromise HIV control in low-income and middle-income countries (LMICs). We aimed to assess the scale of this problem and whether it is associated with the intiation or re-initiation of ART in people who have had previous exposure to antiretroviral drugs. METHODS: This study was a systematic review and meta-regression analysis. We assessed regional prevalence of pretreatment drug resistance and risk of pretreatment drug resistance in people initiating ART who reported previous ART exposure. We systematically screened publications and unpublished datasets for pretreatment drug-resistance data in individuals in LMICs initiating or re-initiating first-line ART from LMICs. We searched for studies in PubMed and Embase and conference abstracts and presentations from the Conference on Retroviruses and Opportunistic Infections, the International AIDS Society Conference, and the International Drug Resistance Workshop for the period Jan 1, 2001, to Dec 31, 2016. To assess the prevalence of drug resistance within a specified region at any specific timepoint, we extracted study level data and pooled prevalence estimates within the region using an empty logistic regression model with a random effect at the study level. We used random effects meta-regression to relate sampling year to prevalence of pretreatment drug resistance within geographical regions. FINDINGS: We identified 358 datasets that contributed data to our analyses, representing 56 044 adults in 63 countries. Prevalence estimates of pretreatment NNRTI resistance in 2016 were 11·0% (7·5-15·9) in southern Africa, 10·1% (5·1-19·4) in eastern Africa, 7·2% (2·9-16·5) in western and central Africa, and 9·4% (6·6-13·2) in Latin America and the Caribbean. There were substantial increases in pretreatment NNRTI resistance per year in all regions. The yearly increases in the odds of pretreatment drug resistance were 23% (95% CI 16-29) in southern Africa, 17% (5-30) in eastern Africa, 17% (6-29) in western and central Africa, 11% (5-18) in Latin America and the Caribbean, and 11% (2-20) in Asia. Estimated increases in the absolute prevalence of pretreatment drug resistance between 2015 and 2016 ranged from 0·3% in Asia to 1·8% in southern Africa. INTERPRETATION: Pretreatment drug resistance is increasing at substantial rate in LMICs, especially in sub-Saharan Africa. In 2016, the prevalence of pretreatment NNRTI resistance was near WHO's 10% threshold for changing first-line ART in southern and eastern Africa and Latin America, underscoring the need for routine national HIV drug-resistance surveillance and review of national policies for first-line ART regimen composition. FUNDING: Bill & Melinda Gates Foundation and World Health Organization

Advances in geometric techniques for analyzing blebbing in chemotaxing Dictyostelium cells.

We present a technical platform that allows us to monitor and measure cortex and membrane dynamics during bleb-based chemotaxis. Using D. discoideum cells expressing LifeAct-GFP and crawling under agarose containing RITC-dextran, we were able to simultaneously visualize the actin cortex and the cell membrane throughout bleb formation. Using these images, we then applied edge detect to generate points on the cell boundary with coordinates in a coordinate plane. Then we fitted these points to a curve with known x and y coordinate functions. The result was to parameterize the cell outline. With the parameterization, we demonstrate how to compute data for geometric features such as cell area, bleb area and edge curvature. This allows us to collect vital data for the analysis of blebbing

A role for myosin II clusters and membrane energy in cortex rupture for Dictyostelium discoideum.

Blebs, pressure driven protrusions of the cell membrane, facilitate the movement of eukaryotic cells such as the soil amoeba Dictyostelium discoideum, white blood cells and cancer cells. Blebs initiate when the cell membrane separates from the underlying cortex. A local rupture of the cortex, has been suggested as a mechanism by which blebs are initiated. However, much clarity is still needed about how cells inherently regulate rupture of the cortex in locations where blebs are expected to form. In this work, we examine the role of membrane energy and the motor protein myosin II (myosin) in facilitating the cell driven rupture of the cortex. We perform under-agarose chemotaxis experiments, using Dictyostelium discoideum cells, to visualize the dynamics of myosin and calculate changes in membrane energy in the blebbing region. To facilitate a rapid detection of blebs and analysis of the energy and myosin distribution at the cell front, we introduce an autonomous bleb detection algorithm that takes in discrete cell boundaries and returns the coordinate location of blebs with its shape characteristics. We are able to identify by microscopy naturally occurring gaps in the cortex prior to membrane detachment at sites of bleb nucleation. These gaps form at positions calculated to have high membrane energy, and are associated with areas of myosin enrichment. Myosin is also shown to accumulate in the cortex prior to bleb initiation and just before the complete disassembly of the cortex. Together our findings provide direct spatial and temporal evidence to support cortex rupture as an intrinsic bleb initiation mechanism and suggests that myosin clusters are associated with regions of high membrane energy where its contractile activity leads to a rupture of the cortex at points of maximal energy

Erratum to: Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition) (Autophagy, 12, 1, 1-222, 10.1080/15548627.2015.1100356

non present

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field