Telomere shortening in cells with low intrinsic telomerase activity like fibroblasts is governed by various mechanisms including the so-called end-replication problem, end processing and oxidative ...DNA damage. To assess the impact of oxidative stress on telomere shortening rates, we compared telomere shortening rates measured in fibroblasts from two different donor species (human and sheep) under both pro- and antioxidative culture regimes. Over an almost 50-fold change in peroxide indicator dye fluorescence intensity, we found a continuous, exponential correlation between cellular oxidative stress levels and telomere shortening rates, which was independent of donor species and cell strain. This correlation suggests stress-mediated telomere DNA damage as an important determinant of telomere shortening.
•Ecoacoustics can increase time and cost efficiency in biodiversity assessments.•We applied four acoustic indices to recordings from an agricultural landscape in Madagascar.•Three acoustic indices ...showed systematic variation among predominant land-use types.•We found a positive correlation of acoustic indices and bird species richness.•High indices in structurally diverse vanilla agroforests suggest a high value for conservation.
New technologies like ecoacoustic surveys promise time and cost efficiency for biodiversity assessments, serve as a basis for effective conservation policies, and are particularly appealing for remote and highly diverse tropical areas. Acoustic indices facilitate the analysis of large acoustic datasets but no consensus on their performance has been reached yet. We evaluated the efficacy of four acoustic indices (Acoustic Complexity Index, Acoustic Diversity Index, Acoustic Evenness Index, Acoustic Entropy) for sound data analysis and biodiversity assessments inside a national park and the agricultural mosaic landscape of north-eastern Madagascar, a global biodiversity hotspot. We used self-built sound recorders to continuously record soundscapes on 80plots across seven land-use types (old-growth forest, forest fragment, forest–derived and fallow-derived vanilla agroforest, herbaceous and woody fallow, rice paddy) and compared index values between land–use types, assessed the correlation with bird species richness as measured by point counts, and related the acoustic indices to plot- and landscape-scale parameters. The Acoustic Diversity Index, Acoustic Evenness Index (inverse) and Acoustic Entropy were highest in old-growth forest and lowest for rice paddies and fallow land. Index values for structurally similar land-use types did not differ significantly from each other. The correlation of the three acoustic indices with bird species richness was strongest during daytime (R2≥0.30). Differences in the index values were best explained by land-use type and vegetation density. Our results showed that all investigated indices except the Acoustic Complexity Index were suitable biodiversity indicators for a tropical, agricultural landscape. Soundscape diversity was positively affected by plot-scale vegetation structure, emphasizing the importance of forests and particularly old-growth forest for conservation. We demonstrated that acoustic indices and sound recordings are a useful tool for assessing biodiversity in tropical agricultural mosaic landscapes. To realize the full potential of ecoacoustics in conservation, sampling guidelines and user-friendly analysis packages will be key to facilitate a wider implementation.
Cellular senescence—the permanent arrest of cycling in normally proliferating cells such as fibroblasts—contributes both to age‐related loss of mammalian tissue homeostasis and acts as a tumour ...suppressor mechanism. The pathways leading to establishment of senescence are proving to be more complex than was previously envisaged. Combining in‐silico interactome analysis and functional target gene inhibition, stochastic modelling and live cell microscopy, we show here that there exists a dynamic feedback loop that is triggered by a DNA damage response (DDR) and, which after a delay of several days, locks the cell into an actively maintained state of ‘deep’ cellular senescence. The essential feature of the loop is that long‐term activation of the checkpoint gene CDKN1A (p21) induces mitochondrial dysfunction and production of reactive oxygen species (ROS) through serial signalling through GADD45‐MAPK14(p38MAPK)‐GRB2‐TGFBR2‐TGFβ. These ROS in turn replenish short‐lived DNA damage foci and maintain an ongoing DDR. We show that this loop is both necessary and sufficient for the stability of growth arrest during the establishment of the senescent phenotype.
Synopsis
The phenomenon of cellular ‘senescence’—the permanent arrest of division in normally proliferating mammalian cells such as fibroblasts—is thought to be a central component of the ageing process. Senescence contributes both to age‐related loss of tissue homeostasis, as the loss of division capacity leads to impaired cell renewal, and also to protect against cancer, because it acts to block the uncontrolled proliferation of cells that may give rise to a malignant tumour. Replicative senescence is triggered by uncapped telomeres or by ‘unrepairable’ non‐telomeric DNA damage. Both lesions initiate the same canonical DNA damage response (DDR) (d'Adda di Fagagna, 2008). This response is characterized by activation of sensor kinases (ATM/ATR, DNA‐PK), formation of DNA damage foci containing activated H2A.X (γH2A.X) and ultimately induction of cell cycle arrest through activation of checkpoint proteins, notably p53 (TP53) and the CDK inhibitor p21 (CDKN1A). This signalling pathway continues to contribute actively to the stability of the G0 arrest in fully senescent cells long after induction of senescence (d'Adda di Fagagna et al, 2003). However, senescence is more complex than mere CDKI‐mediated growth arrest. Senescent cells alter their expression of literally hundreds of genes (Shelton et al, 1999), prominent among these being pro‐inflammatory secretory genes (Coppe et al, 2008) and marker genes for a retrograde response induced by mitochondrial dysfunction (Passos et al, 2007a).
There is a growing evidence that multiple mechanisms interact to underpin ageing at the cellular level (Kirkwood, 2005; Passos et al, 2007b) necessitating a systems biology approach if the complex mechanisms of ageing are to be understood (Kirkwood, 2008). With respect to cell senescence, the two major unanswered questions are (i) How does a DNA lesion that can be repaired, at least in principle, induce and maintain irreversible growth arrest? and (ii) How does a growth arrest trigger a completely different cellular phenotype as soon as it becomes irreversible?
To understand those questions, we performed a kinetic analysis of the establishment phase of senescence initiated by DNA damage or telomere dysfunction, focussing on pathways downstream of the classical DDR. Using an approach that combined (i) in‐silico interactome analysis, (ii) functional target gene inhibition, (iii) stochastic modelling, and (iv) live cell microscopy, we identified a positive feedback loop between DDR and mitochondrial production of reactive oxygen species (ROS) as necessary and sufficient for long‐term maintenance of growth arrest. Using pathway log likelihood scores calculated by a quantitative in‐silico interactome analysis to guide siRNA and small molecule inhibition experiments, and using results of sequential and combined inhibition experiments to refine the predictions from the interactome analysis, we found that DDR triggered mitochondrial dysfunction leading to enhanced ROS activation through a linear signal transduction through TP53, CDKN1A, GADD45A, p38 (MAPK14), GRB2, TGFBR2 and TGFβ(Figure 2D). We hypothesized that these ROS stochastically generate novel DNA damage in the nucleus, thus forming a positive feedback loop contributing to the long‐term maintenance of DDR (Figure 3A). First confirmation came from static inhibitor experiments as before, showing that nuclear DNA damage foci frequencies in senescent cells were reduced if feedback signalling was suppressed. To formally establish the existence of a feedback loop and its relevance for senescence, we used live cell microscopy in combination with quantitative modelling.
We transformed the conceptual model shown in Figure 3A into a stochastic mechanistic model of the DDR feedback loop by extending the previously published model of the TP53/Mdm2 circuit (Proctor and Gray, 2008) to include reactions for synthesis/activation and degradation/deactivation/repair of CDKN1A, GADD45, MAPK14, ROS and DNA damage. The model replicated very precisely the kinetic behaviour of activated TP53, CDKN1A, ROS and DNA damage foci after initiation of senescence by irradiation. Having established its concordance with the experimental data, the model was then used to predict the effects of intervening in the feedback loop. The model predicted that any intervention reducing ROS levels by about half would decrease average DNA damage foci frequencies from six to four foci/nucleus within about 15 h. It further predicted that this would be sufficient to reduce CDKN1A to basal levels continuously for at least 6 h in about 20% of the treated cells, thus allowing a significant fraction of cells to escape from growth arrest and to resume proliferation. This should happen even if the intervention into the feedback loop was started at a late time point (e.g. 6 days) after induction of senescence.
To analyse DNA damage foci dynamics we used a reporter construct (AcGFP–53BP1c) that quantitatively reports single DNA damage foci kinetics in time‐resolved live cell microscopy (Nelson et al, 2009). Foci frequency measurements quantitatively confirmed the prediction from the stochastic model. More importantly, we found that many individual foci in both telomere‐ and stress‐dependent senescence had short lifespans with half‐lives below 15 h. Feedback loop inhibition reduced only the frequencies of short‐lived DNA damage foci in accordance with the hypothesis that ROS production contributed to DDR by constant replenishment of short‐lived DNA damage foci.
Finally, we inhibited signalling through the loop at different time points after induction of senescence by ionizing radiation and measured ROS levels, DNA damage foci frequencies and proliferation markers. Treatments with the MAPK14 inhibitor SB203580 or the free radical scavenger PBN were used to block the loop. The results quantitatively confirmed the model prediction and indicated that the feedback loop between DDR and ROS production was both necessary and sufficient to maintain cell cycle arrest for at least 6–10 days after induction of senescence. Interestingly, the loop was still active at later time points and in deep senescence, but proliferation arrest was then stabilized by additional factor(s). This indicated that certain features of the senescent phenotype‐like ROS production that might be responsible for the negative impact of senescent cells into their tissue environment can be successfully inhibited even in deep senescence. This may prove relevant for novel therapeutic studies aiming to modulate intracellular ROS levels in both aging and cancer.
The sustained activation of CDKN1A (p21/Waf1/Cip1) by a DNA damage response induces mitochondrial dysfunction and reactive oxygen species (ROS) production via signalling through CDKN1A‐GADD45A‐MAPK14‐ GRB2‐TGFBR2‐TGFbeta in senescing primary human and mouse cells in vitro and in vivo.
Enhanced ROS production in senescing cells generates additional DNA damage. Although this damage is repairable and transient, it elevates the average levels of DNA damage response permanently, thus forming a positive feedback loop.
This loop is necessary and sufficient to maintain the stability of growth arrest until a ‘point of no return’ is reached during establishment of senescence.
Aging is an inherently stochastic process, and its hallmark is heterogeneity between organisms, cell types, and clonal populations, even in identical environments. The replicative lifespan of primary ...human cells is telomere dependent; however, its heterogeneity is not understood. We show that mitochondrial superoxide production increases with replicative age in human fibroblasts despite an adaptive UCP-2-dependent mitochondrial uncoupling. This mitochondrial dysfunction is accompanied by compromised Ca(2+)i homeostasis and other indicators of a retrograde response in senescent cells. Replicative senescence of human fibroblasts is delayed by mild mitochondrial uncoupling. Uncoupling reduces mitochondrial superoxide generation, slows down telomere shortening, and delays formation of telomeric gamma-H2A.X foci. This indicates mitochondrial production of reactive oxygen species (ROS) as one of the causes of replicative senescence. By sorting early senescent (SES) cells from young proliferating fibroblast cultures, we show that SES cells have higher ROS levels, dysfunctional mitochondria, shorter telomeres, and telomeric gamma-H2A.X foci. We propose that mitochondrial ROS is a major determinant of telomere-dependent senescence at the single-cell level that is responsible for cell-to-cell variation in replicative lifespan.
The endothelium plays a key role in the dynamic balance of hemodynamic, humoral and inflammatory processes in the human body. Its central importance and the resulting therapeutic concepts are the ...subject of ongoing research efforts and form the basis for the treatment of numerous diseases. The pulmonary endothelium is an essential component for the gas exchange in humans. Pulmonary endothelial dysfunction has serious consequences for the oxygenation and the gas exchange in humans with the potential of consecutive multiple organ failure. Therefore, in this review, the dysfunction of the pulmonary endothel due to viral, bacterial, and fungal infections, ventilator-related injury, and aspiration is presented in a medical context. Selected aspects of the interaction of endothelial cells with primarily alveolar macrophages are reviewed in more detail. Elucidation of underlying causes and mechanisms of damage and repair may lead to new therapeutic approaches. Specific emphasis is placed on the processes leading to the induction of cyclooxygenase-2 and downstream prostanoid-based signaling pathways associated with this enzyme.
Every year, more information accumulates about the possibility of treating patients with acute lung injury or acute respiratory distress syndrome with specially designed mechanical ventilation ...strategies. Ventilator modes, positive end-expiratory pressure settings, and recruitment maneuvers play a major role in these strategies. However, what can we take from these experimental and clinical data to the clinical practice? In this article, we discuss substantial options of mechanical ventilation together with some adjunctive therapeutic measures, such as prone positioning and inhalation of nitric oxide.
Dislocation of catheters within the tissue is a challenge in continuous regional anesthesia. A novel self-coiling catheter design is available and has demonstrated a lower dislocation rate in a ...cadaver model. The dislocation rate and effect on postoperative pain of these catheters in vivo has yet to be determined and were the subjects of this investigation.
After ethics committee approval 140 patients undergoing elective distal lower limb surgery were enrolled in this prospective randomized controlled trial. Preoperatively, patients were randomly assigned and received either the conventional (n = 70) or self-coiling catheter (n = 70) for ultrasound-guided popliteal sciatic nerve block in short axis view and by the in-plane approach from lateral to medial. The primary outcome was pain intensity after surgery and on the following three postoperative days. Secondary outcomes investigated were dislocation rate in situ determined by sonography, catheter movement visible from outside, opioid consumption as well as leakage at the puncture site.
All catheters were successfully inserted. The study population of self-coiling catheters had significantly lower mean numeric rating scale values than the reference cohort on the first (p = 0.01) and second postoperative days (p < 0.01). Sonographic evaluation demonstrated, 42 standard catheters (60%) and 10 self-coiling catheters (14.3%) were dislocated in situ within the first three postoperative days. The externally visible movement of the catheters at insertion site did not differ significantly between groups through the third postoperative day. The opioid consumption was significantly lower in the self-coiling catheter group on the day of surgery and on the second and third postoperative days (p = 0.04, p = 0.03 and p = 0.04, respectively).
The self-coiling catheter offers a better postoperative pain control and a lower dislocation rate within the tissue when blocking the popliteal sciatic nerve compared to a conventional catheter. Further trials in large patient cohorts are warranted to investigate the potential beneficial effects of self-coiling catheters for other localisations and other application techniques.
The trial was registered at German Clinical Trials Register (DRKS) on 08/04/2020 ( DRKS00020938 , retrospectively registered).
Aspiration of low-pH gastric fluid leads to an initial pneumonitis, which may become complicated by subsequent pneumonia or acute respiratory distress syndrome. Current treatment is at best ...supportive, but there is growing experimental evidence on the significant contribution of both neutrophils and platelets in the development of this inflammatory pulmonary reaction, a condition that can be attenuated by several medicinal products. This review aims to summarize novel findings in experimental models on pathomechanisms after an acid-aspiration event. Given the clinical relevance, specific emphasis is put on deduced potential experimental therapeutic approaches, which make use of the characteristic alteration of microcirculation in the injured lung.
The prone position is used to improve gas exchange in patients with acute respiratory distress syndrome. However, the regional mechanism by which the prone position improves gas exchange in acutely ...injured lungs is still incompletely defined.
We used positron emission tomography imaging of (13)Nnitrogen to assess the regional distribution of pulmonary shunt, aeration, perfusion, and ventilation in seven surfactant-depleted sheep in supine and prone positions.
In the supine position, the dorsal lung regions had a high shunt fraction, high perfusion, and poor aeration. The prone position was associated with an increase in lung gas content and with a more uniform distribution of aeration, as the increase in aeration in dorsal lung regions was not offset by loss of aeration in ventral regions. Consequently, the shunt fraction decreased in dorsal regions in the prone position without a concomitant impairment of gas exchange in ventral regions, thus leading to a significant increase in the fraction of pulmonary perfusion participating in gas exchange. In addition, the vertical distribution of specific alveolar ventilation became more uniform in the prone position. A biphasic relation between regional shunt fraction and gas fraction showed low shunt for values of gas fraction higher than a threshold, and a steep linear increase in shunt for lower values of gas fraction.
In a surfactant-deficient model of lung injury, the prone position improved gas exchange by restoring aeration and decreasing shunt while preserving perfusion in dorsal lung regions, and by making the distribution of ventilation more uniform.
Human–wildlife interactions are usually centred on the conflict between local populations and species that are perceived as problematic. To better understand the human dimension, social science ...approaches are increasingly incorporated to delimit opportunities for coexistence and species conservation.
Here we explore local attitudes and beliefs about one of these ‘problematic’ species, the Endangered Aye‐aye Daubentonia madagascariensis, the largest nocturnal primate on earth. We conducted a literature review on published beliefs and narratives about the Aye‐aye followed by 83 semi‐structured interviews in 11 villages in northeastern Madagascar.
The Aye‐aye is generally perceived as a bad omen throughout its range. In many places it has to be killed on sight, while in others it is forbidden to be killed. We did not find any positive attitudes towards this species in the literature. However, this was not reflected by our interviews: although 47.0% of respondents held a negative attitude, more than half had a neutral or even positive attitude towards the Aye‐aye (36.1 and 18.1% respectively). Positive attitudes were linked to perceived pest control services on major cash crops. Negative attitudes are mostly related to the perception that when an Aye‐aye comes to a village, catastrophic things (e.g. deaths of community members) will happen, or that it destroys a village. These major narratives were mostly consistent within villages (79.5 ± 20.7% SD of respondents; range 50–100%) but considerably differed between villages.
Negative attitudes towards the Aye‐aye were solely based on vague and generic narratives that are not reflected by its (true) ecology. Positive and neutral attitudes instead were related to observable behaviours benefitting people (i.e. pest control services) or curiosity about the species. This case study illustrates how even firm narratives can vary locally offering valuable starting points for targeted conservation or education programs. We therefore identified the Makira region of northeastern Madagascar as a potential stronghold for Aye‐aye conservation if specific actions highlighting its beneficial value could be initiated. In general, conservation practitioners should feel encouraged to look beyond the often told stories about ‘their’ target species and listen to local voices more often.
Résumé
Les interactions entre l'homme et la faune sauvage sont généralement focalisées sur le conflit entre les populations locales et les espèces qui sont reconnues comme problématiques. Pour mieux comprendre la dimension humaine, les approches des sciences sociales sont de plus en plus intégrées pour délimiter de nouvelles possibilités de coexistence et de conservation des espèces.
Nous explorons dans le présent document les attitudes et les croyances locales concernant l'une de ces espèces ‘problématiques’, l'Aye‐aye (Daubentonia madagascariensis), le plus grand primate nocturne de la planète, qui est en voie de disparition. Nous avons effectué une analyse documentaire sur les croyances et les récits publiés au sujet de l'Aye‐aye, suivie de 83 entretiens dans onze villages, au Nord‐est de Madagascar.
L'Aye‐aye est généralement considéré comme un mauvais augure dans toute son aire de répartition. Dans de nombreux endroits, il doit être abattu dès qu'il est aperçu, alors que dans d'autres, il est interdit de le tuer. Nous n'avons trouvé aucune attitude positive envers cette espèce dans la littérature. Cependant, cela ne se reflète pas dans nos entretiens: bien que 47,0% des personnes interrogées aient une attitude négative, plus de la moitié ont une attitude neutre ou même positive envers l'Aye‐aye (36,1 et 18,1%, respectivement). Les attitudes positives étaient liées à la perception des services de lutte contre les parasites sur les principales cultures de rente. Les attitudes négatives sont principalement liées à la croyance que lorsqu'un Aye‐aye vient dans un village, des événements catastrophiques (par exemple, la mort de membres de la communauté) vont se produire ou il va détruire le village. Ces principaux récits étaient pour la plupart récurrents au sein des villages (79,5 ± 20,7% ET des personnes enquêtées; entre 50 et 100%) mais différaient considérablement d'un village à l'autre.
Les attitudes négatives envers l'Aye‐aye étaient uniquement basées sur des récits vagues et habituels qui ne sont pas reflétés par son (véritable) écologie. Les attitudes positives et neutres étaient plutôt liées à des actions visibles qui sont bénéfiques pour les gens (par exemple, les services de lutte contre les parasites) ou à la curiosité vis‐à‐vis de l'espèce. Cette analyse illustre la manière dont même les propos fermes sur certaines espèces peuvent varier localement, offrant ainsi des points de départ précieux pour des programmes ciblés de conservation ou d'éducation. Nous avons donc identifié la région de Makira, au Nord‐est de Madagascar, comme un fief potentiel pour la conservation de l'Aye‐aye si des actions spécifiques mettant en évidence sa valeur bénéfique pouvaient être initiées. En général, les praticiens de la conservation devraient se sentir encouragés à examiner au‐delà des histoires souvent racontées sur ‘leurs’ espèces cibles et à écouter plus souvent les voix locales.
A free Plain Language Summary can be found within the Supporting Information of this article.
A free Plain Language Summary can be found within the Supporting Information of this article.
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