Telomere length is an epigenetic trait - Implications for the use of telomerase-deficient organisms to model human disease.

Telomere length, unlike most genetic traits, is epigenetic, in the sense that it is not fully coded by the genome. Telomeres vary in length and randomly assort to the progeny leaving some individuals with longer and others with shorter telomeres. Telomerase activity counteracts this by extending telomeres in the germline and during embryogenesis but sizeable variances remain in telomere length. This effect is exacerbated by the absence of fully active telomerase. Telomerase heterozygous animals (tert+/-) have reduced telomerase activity and their telomeres fail to be elongated to wild-type average length, meaning that - with every generation - they decrease. After a given number of successive generations of telomerase-insufficient crosses, telomeres become critically short and cause organismal defects that, in humans, are known as telomere biology disorders. Importantly, these defects also occur in wild-type (tert+/+) animals derived from such tert+/- incrosses. Despite these tert+/+ animals being proficient for telomerase, they have shorter than average telomere length and, although milder, develop phenotypes that are similar to those of telomerase mutants. Here, we discuss the impact of this phenomenon on human pathologies associated with telomere length, provide a brief overview of telomere biology across species and propose specific measures for working with telomerase-deficient zebrafish.

Adjuvant Botulinum Toxin Type A on the Management of Giant Hiatal Hernia: A Case Report.

The management of giant hiatal hernias (HHs) remains challenging and is associated with a high risk of recurrence. Currently, several strategies are used to reduce recurrence, and a newly proposed trend is the administration of adjuvant botulinum toxin type A (BTX), a procedure already performed in complex ventral hernias. Here, we present a case of a 63-year-old man with a giant paraesophageal HH type IV containing the entire stomach and transverse colon with loss of domain, who underwent adjuvant BTX and subsequently laparoscopic hiatoplasty with a biological mesh with partial fundoplication. At six months' follow-up, the patient reported a significant improvement in the quality of life without dysphagia or gastroesophageal reflux and with a good respiratory function. A control computed tomography was performed, which documented a partial recurrence of HH, completely asymptomatic. This clinical case showed the successful treatment of a giant HH using adjuvant BTX injection to increase abdominal wall compliance as had already been described in the treatment of complex ventral hernia. Thus, the use of BTX is a promising strategy for selected cases of giant HHs mainly if there is a loss of domain; however, more case series and controlled trials are needed to show the reproducibility of the benefit of this strategy.

Lead to hit ruthenium-cyclopentadienyl anticancer compounds: Cytotoxicity against breast cancer cells, metabolic stability and metabolite profiling.

The successful choice of hit compounds during drug development programs involves the integration of structure-activity relationship (SAR) studies with pharmacokinetic determinations, including metabolic stability assays and metabolite profiling. A panel of nine ruthenium-cyclopentadienyl (RuCp) compounds with the general formula [Ru(η5-C5H4R)(PPh3)(bipyR')]+ (with R = H, CHO, CH2OH; R' = H, CH3, CH2OH, CH2Biotin) has been tested against hormone-dependent MCF-7 and triple negative MDA-MB-231 breast cancer cells. In general, all compounds showed important cytotoxicity against both cancer cell lines and were able to inhibit the formation of MDA-MB-231 colonies in a dose-dependent manner, while showing selectivity for cancer cells over normal fibroblasts. Among them, four compounds stood out as lead structures to be further studied. Cell distribution assays revealed their preference for the accumulation at cell membrane (Ru quantification by ICP-MS) and the mechanism of cell death seemed to be mediated by apoptosis. Potential structural liabilities of lead compounds were subsequently flagged upon in vitro metabolic stability assays and metabolite profiling. The implementation of this integrated strategy led to the selection of RT151 as a promising hit compound.

Vivências de Violência e Percepção do Medo entre Estudantes Universitários / Experiences of Violence and Perception of Fear among University Students / Experiencias de Violencia y Percepción del Miedo entre Estudiantes Universitarios

O objetivo deste estudo foi traçar um panorama das violências vivenciadas e temidas por estudantes no contexto universitário. Buscou-se descrever as frequências dessas violências, bem como a percepção dos alunos sobre suporte psicológico da Universidade e denúncia do agressor. Os dados foram obtidos por meio de questionário online e sistematizados com o auxílio do software SPSS (versão 2.3). Utilizou-se análises descritivas de frequência, média, desvio padrão, quartis e teste-t. Em geral, as médias de vivência de violência foram baixas, com maior incidência de violência emocional de professor e de colega; mulheres disseram ter sofrido mais violência em relação aos homens. As médias do medo foram altas, especialmente para mulheres, alunos mais jovens, com menor renda salarial e que relataram já ter sofrido algum tipo de violência na universidade. A universidade se mostrou um local onde parece ocorrer menos violência do que se teme. Estratégias de ação das universidades são necessárias para uma cultura solidária entre estudantes, professores e funcionários e para a oferta de suporte psicológico aos estudantes.

This study aimed to provide an overview of the violence experienced and feared by students in the university context. We describe violence frequencies, students' perception of psychological support provided by the University, and the rate of denouncement of the aggressor. The data were obtained through an online questionnaire and systematized in the SPSS software (version 2.3). Descriptive analyzes of frequency, mean, standard deviation, quartiles, and t-test were used. Overall, the average experience of violence was low, with a higher incidence of emotional violence by teachers and colleagues; women reported suffering more violence than men. The averages of reported fear were high, especially for women, younger, and lower-income students, and those who reported having suffered some type of violence at the university. The university proved to be a place where there seems to be less violence than is feared. Action strategies from universities are necessary for a solidary culture among students, teachers, and staff and for offering psychological support to students.

El objetivo de este estudio fue esbozar un panorama de la violencia vivida y temida por los estudiantes en el contexto universitario. Se buscó describir las frecuencias de estas violencias, así como la percepción de los estudiantes sobre el apoyo psicológico de la Universidad y la denuncia del agresor. Los datos se obtuvieron a través de un cuestionario en línea y se sistematizaron con la ayuda del software SPSS (versión 2.3). Se utilizaron análisis descriptivos de frecuencia, media, desviación estándar, cuartiles y prueba t. En general, la experiencia media de violencia fue baja, con mayor incidencia de violencia emocional por parte de profesores y compañeros; las mujeres dijeron que sufrieron más violencia que los hombres. Los promedios de miedo fueron altos, especialmente para las mujeres, estudiantes más jóvenes, con menores ingresos y que reportaron haber sufrido algún tipo de violencia en la universidad. La universidad resultó ser un lugar donde parece haber menos violencia de la que se teme. Las estrategias de acción de las universidades son necesarias para una cultura solidaria en ese contexto y para ofrecer apoyo psicológico a los estudiantes.

A p21-GFP zebrafish model of senescence for rapid testing of senolytics in vivo.

Senescence drives the onset and severity of multiple ageing-associated diseases and frailty. As a result, there has been an increased interest in mechanistic studies and in the search for compounds targeting senescent cells, known as senolytics. Mammalian models are commonly used to test senolytics and generate functional and toxicity data at the level of organs and systems, yet this is expensive and time consuming. Zebrafish share high homology in genes associated with human ageing and disease. They can be genetically modified relatively easily. In larvae, most organs develop within 5 days of fertilisation and are transparent, which allows tracking of fluorescent cells in vivo in real time, testing drug off-target toxicity and assessment of cellular and phenotypic changes. Here, we have generated a transgenic zebrafish line that expresses green fluorescent protein (GFP) under the promoter of a key senescence marker, p21. We show an increase in p21:GFP+ cells in larvae following exposure to ionising radiation and with natural ageing. p21:GFP+ cells display other markers of senescence, including senescence-associated ß-galactosidase and IL6. The observed increase in senescent cells following irradiation is associated with a reduction in the thickness of muscle fibres and mobility, two important ageing phenotypes. We also show that quercetin and dasatinib, two senolytics currently in clinical trials, reduce the number of p21:GFP+ cells, in a rapid 5-day assay. This model provides an important tool to study senescence in a living organism, allowing the rapid selection of senolytics before moving to more expensive and time-consuming mammalian systems.

A subset of gut leukocytes has telomerase-dependent "hyper-long" telomeres and require telomerase for function in zebrafish.

BACKGROUND: Telomerase, the enzyme capable of elongating telomeres, is usually restricted in human somatic cells, which contributes to progressive telomere shortening with cell-division and ageing. T and B-cells cells are somatic cells that can break this rule and can modulate telomerase expression in a homeostatic manner. Whereas it seems intuitive that an immune cell type that depends on regular proliferation outbursts for function may have evolved to modulate telomerase expression it is less obvious why others may also do so, as has been suggested for macrophages and neutrophils in some chronic inflammation disease settings. The gut has been highlighted as a key modulator of systemic ageing and is a key tissue where inflammation must be carefully controlled to prevent dysfunction. How telomerase may play a role in innate immune subtypes in the context of natural ageing in the gut, however, remains to be determined. RESULTS: Using the zebrafish model, we show that subsets of gut immune cells have telomerase-dependent"hyper-long" telomeres, which we identified as being predominantly macrophages and dendritics (mpeg1.1+ and cd45+mhcII+). Notably, mpeg1.1+ macrophages have much longer telomeres in the gut than in their haematopoietic tissue of origin, suggesting that there is modulation of telomerase in these cells, in the gut. Moreover, we show that a subset of gut mpeg1.1+ cells express telomerase (tert) in young WT zebrafish, but that the relative proportion of these cells decreases with ageing. Importantly, this is accompanied by telomere shortening and DNA damage responses with ageing and a telomerase-dependent decrease in expression of autophagy and immune activation markers. Finally, these telomerase-dependent molecular alterations are accompanied by impaired phagocytosis of E. coli and increased gut permeability in vivo. CONCLUSIONS: Our data show that limiting levels of telomerase lead to alterations in gut immunity, impacting on the ability to clear pathogens in vivo. These are accompanied by increased gut permeability, which, together, are likely contributors to local and systemic tissue degeneration and increased susceptibility to infection with ageing.

Müller Glia maintain their regenerative potential despite degeneration in the aged zebrafish retina.

Ageing is a significant risk factor for degeneration of the retina. Müller glia cells (MG) are key for neuronal regeneration, so harnessing the regenerative capacity of MG in the retina offers great promise for the treatment of age-associated blinding conditions. Yet, the impact of ageing on MG regenerative capacity is unclear. Here, we show that the zebrafish retina undergoes telomerase-independent, age-related neurodegeneration but that this is insufficient to stimulate MG proliferation and regeneration. Instead, age-related neurodegeneration is accompanied by MG morphological aberrations and loss of vision. Mechanistically, yes-associated protein (Yap), part of the Hippo signalling, has been shown to be critical for the regenerative response in the damaged retina, and we show that Yap expression levels decline with ageing. Despite this, morphologically and molecularly altered aged MG retain the capacity to regenerate neurons after acute light damage, therefore, highlighting key differences in the MG response to high-intensity acute damage versus chronic neuronal loss in the zebrafish retina.

Violência emocional intrafamiliar contra crianças e adolescentes e suas repercussões: uma revisão sistemática de literatura / Repercussions of emotional family violence against children and adolescents: a systematic review / Repercusiones de la violencia emocional intrafamiliar contra niños y adolescentes: una revisión sistemática

O objetivo deste estudo foi identificar as repercussões na infância, na adolescência e na vida adulta da violência emocional intrafamiliar vivenciada no período infanto-juvenil em artigos publicados entre 2009 e 2019, por meio de uma revisão sistemática de literatura. Para a organização dos dados, utilizou-se o software Zotero; para a sistematização e análises, o software SPSS (versão 2.3). Na infância e na adolescência, as principais repercussões foram sintomas internalizantes (ex.: ansiedade, depressão, problemas psicossomáticos, insegurança), problemas cognitivo-comportamentais e impactos no rendimento escolar. Na idade adulta, as principais repercussões foram sintomas externalizantes (ex.: agressividade, reprodução da violência) e sintomas internalizantes. Conclui-se que a violência emocional vivenciada na infância e na adolescência é transcultural, transgeracional e um fator de risco para o desenvolvimento, podendo ocasionar prejuízos cognitivo-comportamentais e interferir nos relacionamentos interpessoais ao longo do ciclo de vida. Tornar visível este tipo de violência contribui para enfrentamento e minimização dos efeitos negativos da violência.

This study aimed to identify the repercussions in childhood, adolescence and adulthood of intrafamily emotional violence experienced in children and adolescents in articles published between 2009 and 2019 through a systematic literature review. To organize the data, the Zotero software was used; for systematization and analysis assistance, the SPSS software (version 2.3). In childhood and adolescence, the main repercussions were internalizing symptoms (for instance: anxiety, depression), cognitive-behavioural problems and impacts on school performance. In adulthood, the main repercussions were externalizing symptoms (for instance: aggressiveness, reproduction of violence) and internalizing symptoms. It follows that the emotional violence experienced in childhood is cross-cultural, transgenerational, and a risk factor for development, as it can cause cognitive-behavioural losses and interfere in interpersonal relationships throughout the life cycle. Making this type of violence visible helps to face and minimize the negative effects of violence.

El objetivo de esta revisión sistemática de la literatura fue identificar las repercusiones en la infancia, adolescencia y vida adulta de la violencia emocional intrafamiliar experimentada en niños y adolescentes en artículos publicados entre 2009 y 2019. Para organizar los datos, se utilizó el software Zotero; para la sistematización y análisis, el software SPSS (versión 2.3). En la infancia y la adolescencia, las principales repercusiones fueron los síntomas internos (ansiedad, depresión), problemas cognitivo-conductuales e impactos en el rendimiento escolar. En la edad adulta, las principales repercusiones fueron síntomas externos (agresividad, reproducción de violencia) y síntomas internos. La violencia emocional experimentada en la infancia es transcultural, transgeneracional y un factor de riesgo para el desarrollo, ya que puede causar impedimentos cognitivo-conductuales e interferir en las relaciones interpersonales a lo largo del ciclo de vida. Hacer visible este tipo de violencia ayuda a enfrentar y minimizar los efectos negativos de la violencia.

Resident Immunity in Tissue Repair and Maintenance: The Zebrafish Model Coming of Age.

The zebrafish has emerged as an exciting vertebrate model to study different aspects of immune system development, particularly due to its transparent embryonic development, the availability of multiple fluorescent reporter lines, efficient genetic tools and live imaging capabilities. However, the study of immunity in zebrafish has largely been limited to early larval stages due to an incomplete knowledge of the full repertoire of immune cells and their specific markers, in particular, a lack of cell surface antibodies to detect and isolate such cells in living tissues. Here we focus on tissue resident or associated immunity beyond development, in the adult zebrafish. It is our view that, with our increasing knowledge and the development of improved tools and protocols, the adult zebrafish will be increasingly appreciated for offering valuable insights into the role of immunity in tissue repair and maintenance, in both health and disease throughout the lifecourse.

Generation of Gellan Gum-Based Adipose-Like Microtissues.

Adipose tissue is involved in many physiological processes. Therefore, the need for adipose tissue-like analogues either for soft tissue reconstruction or as in vitro testing platforms is undeniable. In this work, we explored the natural features of gellan gum (GG) to recreate injectable stable adipose-like microtissues. GG hydrogel particles with different percentages of polymer (0.5%, 0.75%, 1.25%) were developed and the effect of obtained mechanical properties over the ability of hASCs to differentiate towards the adipogenic lineage was evaluated based on the expression of the early (PPARγ) and late (FABP4) adipogenic markers, and on lipids formation and accumulation. Constructs were cultured in adipogenic induction medium up to 21 days or for six days in induction plus nine days in maintenance media. Overall, no significant differences were observed in terms of hASCs adipogenic differentiation within the range of Young’s moduli between 2.7 and 12.9 kPa. The long-term (up to six weeks) stability of the developed constructs supported its application in soft tissue reconstruction. Moreover, their ability to function as adipose-like microtissue models for drug screening was demonstrated by confirming its sensitivity to TNFα and ROCK inhibitor, respectively involved in the repression and induction of the adipogenic differentiation.

How to Catch a Smurf? - Ageing and Beyond In vivo Assessment of Intestinal Permeability in Multiple Model Organisms.

The Smurf Assay (SA) was initially developed in the model organism Drosophila melanogaster where a dramatic increase of intestinal permeability has been shown to occur during aging (Rera et al., 2011). We have since validated the protocol in multiple other model organisms (Dambroise et al., 2016) and have utilized the assay to further our understanding of aging (Tricoire and Rera, 2015; Rera et al., 2018). The SA has now also been used by other labs to assess intestinal barrier permeability (Clark et al., 2015; Katzenberger et al., 2015; Barekat et al., 2016; Chakrabarti et al., 2016; Gelino et al., 2016). The SA in itself is simple; however, numerous small details can have a considerable impact on its experimental validity and subsequent interpretation. Here, we provide a detailed update on the SA technique and explain how to catch a Smurf while avoiding the most common experimental fallacies.

The Zebrafish as an Emerging Model to Study DNA Damage in Aging, Cancer and Other Diseases.

Cancer is a disease of the elderly, and old age is its largest risk factor. With age, DNA damage accumulates continuously, increasing the chance of malignant transformation. The zebrafish has emerged as an important vertebrate model to study these processes. Key mechanisms such as DNA damage responses and cellular senescence can be studied in zebrafish throughout its life course. In addition, the zebrafish is becoming an important resource to study telomere biology in aging, regeneration and cancer. Here we review some of the tools and resources that zebrafish researchers have developed and discuss their potential use in the study of DNA damage, cancer and aging related diseases.

Correction: Telomerase Is Required for Zebrafish Lifespan.

[This corrects the article DOI: 10.1371/journal.pgen.1003214.].

Interventions for age-related diseases: Shifting the paradigm.

Over 60% of people aged over 65 are affected by multiple morbidities, which are more difficult to treat, generate increased healthcare costs and lead to poor quality of life compared to individual diseases. With the number of older people steadily increasing this presents a societal challenge. Age is the major risk factor for age-related diseases and recent research developments have led to the proposal that pharmacological interventions targeting common mechanisms of ageing may be able to delay the onset of multimorbidity. Here we review the state of the knowledge of multimorbidity, appraise the available evidence supporting the role of mechanisms of ageing in the development of the most common age-related diseases and assess potential molecules that may successfully target those key mechanisms.

Short Telomeres in Key Tissues Initiate Local and Systemic Aging in Zebrafish.

Telomeres shorten with each cell division and telomere dysfunction is a recognized hallmark of aging. Tissue proliferation is expected to dictate the rate at which telomeres shorten. We set out to test whether proliferative tissues age faster than non-proliferative due to telomere shortening during zebrafish aging. We performed a prospective study linking telomere length to tissue pathology and disease. Contrary to expectations, we show that telomeres shorten to critical lengths only in specific tissues and independently of their proliferation rate. Short telomeres accumulate in the gut but not in other highly proliferative tissues such as the blood and gonads. Notably, the muscle, a low proliferative tissue, accumulates short telomeres and DNA damage at the same rate as the gut. Together, our work shows that telomere shortening and DNA damage in key tissues triggers not only local dysfunction but also anticipates the onset of age-associated diseases in other tissues, including cancer.

Telomerase is required for zebrafish lifespan.

Telomerase activity is restricted in humans. Consequentially, telomeres shorten in most cells throughout our lives. Telomere dysfunction in vertebrates has been primarily studied in inbred mice strains with very long telomeres that fail to deplete telomeric repeats during their lifetime. It is, therefore, unclear how telomere shortening regulates tissue homeostasis in vertebrates with naturally short telomeres. Zebrafish have restricted telomerase expression and human-like telomere length. Here we show that first-generation tert(-/-) zebrafish die prematurely with shorter telomeres. tert(-/-) fish develop degenerative phenotypes, including premature infertility, gastrointestinal atrophy, and sarcopaenia. tert(-/-) mutants have impaired cell proliferation, accumulation of DNA damage markers, and a p53 response leading to early apoptosis, followed by accumulation of senescent cells. Apoptosis is primarily observed in the proliferative niche and germ cells. Cell proliferation, but not apoptosis, is rescued in tp53(-/-)tert(-/-) mutants, underscoring p53 as mediator of telomerase deficiency and consequent telomere instability. Thus, telomerase is limiting for zebrafish lifespan, enabling the study of telomere shortening in naturally ageing individuals.

Consequences of telomere shortening during lifespan.

Telomerase expression is restricted in human cells and so telomeres shorten throughout our lives, providing a tumour suppressor mechanism that limits cell proliferation. As a trade-off, continuous telomere erosion results in replicative senescence and contributes to ageing. Recently, telomerase therapies were proposed as a valid approach to rescue degenerative phenotypes caused by telomere dysfunction. However, systemic effects initiated by short telomeres may prove dominant in limiting tissue renewal in the whole organism. Most of our knowledge of telomere biology derives from mouse models that do not rely on telomere exhaustion for controlling cell proliferation and tissue homeostasis. In order to understand the impact of telomere shortening in natural ageing, we need to investigate animal models that, like humans, have evolved to have telomere length as a cell division clock.

IL-7 induces rapid clathrin-mediated internalization and JAK3-dependent degradation of IL-7Ralpha in T cells.

Interleukin-7 (IL-7) is an essential cytokine for T-cell development and homeostasis. It is well established that IL-7 promotes the transcriptional down-regulation of IL7RA, leading to decreased IL-7Ralpha surface expression. However, it is currently unknown whether IL-7 regulates the intracellular trafficking and early turnover of its receptor on ligand binding. Here, we show that, in steady-state T cells, IL-7Ralpha is slowly internalized and degraded while a significant fraction recycles back to the surface. On IL-7 stimulation, there is rapid IL-7Ralpha endocytosis via clathrin-coated pits, decreased receptor recycling, and accelerated lysosome and proteasome-dependent degradation. In accordance, the half-life of IL-7Ralpha decreases from 24 hours to approximately 3 hours after IL-7 treatment. Interestingly, we further demonstrate that clathrin-dependent endocytosis is necessary for efficient IL-7 signal transduction. In turn, pretreatment of T cells with JAK3 or pan-JAK inhibitors suggests that IL-7Ralpha degradation depends on the activation of the IL-7 signaling effector JAK3. Overall, our findings indicate that IL-7 triggers rapid IL-7Ralpha endocytosis, which is required for IL-7-mediated signaling and subsequent receptor degradation.

Centrifugal elutriation as a means of cell cycle phase separation and synchronisation.

The ability to purify cells in specific phases of the cell cycle in sufficient quantities for biochemical analysis or to obtain a "synchronized" population of living cells enriched in a particular phase is of great value for studying a wide range of cell cycle processes. Transformed tumour cell lines, such as DT40, can be arrested and released from specific points in the cell cycle using drugs which inhibit DNA or protein synthesis or interfere with mitotic spindle function, however such drugs are frequently toxic and may themselves perturb the cell cycle process or phenomenon under study. Centrifugal elutriation provides a means of separating an unperturbed culture of living cells into highly enriched G1-, S-, and G2/ M-phase fractions. The resulting cell fractions can be analysed directly for protein and mRNA expression or returned to culture and their subsequent progression through the cell cycle monitored by flow cytometry, microscopy, or other techniques.