Poecilia reticulata) brain size selection lines with a pronounced negative association between brain and gut size and investigated feeding propensity, digestive efficiency (DE), and juvenile growth rate. We did not find any difference in feeding propensity or DE between large‐ and small‐brained individuals. Instead, we found that large‐brained females had slower growth during the first 10 weeks after birth. Our study provides experimental support that investment into larger brains at the expense of gut tissue carries costs that are not necessarily compensated by a more efficient digestive system.One key hypothesis in the study of brain size evolution is the expensive tissue hypothesis; the idea that increased investment into the brain should be compensated by decreased investment into other costly organs, for instance the gut. Although the hypothesis is supported by both comparative and experimental evidence, little is known about the potential changes in energetic requirements or digestive traits following such evolutionary shifts in brain and gut size. Organisms may meet the greater metabolic requirements of larger brains despite smaller guts via increased food intake or better digestion. But increased investment in the brain may also hamper somatic growth. To test these hypotheses we here used guppy ( In these lines, adult body size was not different, but relative (and absolute) brain size was 9% larger in the up‐selected lines already after two generations of selection. In support of the expensive tissue hypothesis, the difference in brain size was accompanied by substantial differences also in gut size. Gut size was substantially smaller  in large brained lines than in small brained lines  do not compensate for a smaller gut. For instance, the most critical phase of human brain development is offset by reduced growth in early childhood as compared to other primates with smaller brains  800  and 100  sinking food pellets , which could be consumed in one bite. Feeding usually stopped after 4 min. After 30 min, we removed and counted all uneaten pellets (pellets did not disintegrate). The 120 individuals in this experiment were kept in 12 tanks and assayed for 10 days. The number of observations was therefore 120.Digestive assimilation efficiency is the ratio of energy assimilated to energy ingested . Ninety‐three milliliters of ultrapure water was added, and the solution was filtered (0.45 μm), diluted 1:10 with 3% nitric acid, Co was added as internal standard, and Cr was analyzed by ICP‐CRI‐MS (inductively coupled plasma collision/reaction interface mass spectrometry) with a Varian 820‐MS  applying 80 mL/min hydrogen as collision gas to the sampler cone. The rest of the sample (12 mg [range: 2–28 mg]) was used to determine energy content; we determined energy content (J/g) from food and feces by bomb calorimetry using an adiabatic calorimeter  with a microbomb insert. Following Flowerdew and Grove . Although we collected feces of 180 fish over several days, the analysis requires relatively large sample amounts, which made it necessary to pool samples. The number of calorimetric measurements was therefore 12 .The amount of sample used in the analysis did not affect the calorimetry result  and 3.0  food pellets per feeding bout and individual, but brain size selection regime did not affect food intake  and artificial selection for relative brain size did not influence this efficiency; also males and females showed similar rates of DE .Large‐ and small‐brained guppies kept in individual tanks for two years did not differ in body size , whereas animals from low predation tend to consume more low‐quality food items (detritus and algae; Bassar et al. In our experimental set‐up, food availability was fixed. Animals could therefore not resort to the above‐described behavioral adaptations. This may explain why females of the large‐brained lines displayed slower juvenile growth than females from the small‐brained lines. But this finding also points toward an important net cost of having a larger brain and lends further support for the expensive tissue hypothesis. It is apparent that even under benign feeding conditions, such as those used in our experimental setup, the combination of a large brain and a small gut carries costs that limit juvenile growth. Even though the difference in relative brain size is evident already at birth in the guppy brain size selection lines (Kotrschal et al. Reduced juvenile growth rate may negatively impact large‐brained animals in at least two ways. First, in fish and many other aquatic organisms, mortality usually decreases strongly with increasing body size, because the most important aquatic predators are gape‐size limited Sogard . The larWe did not detect any difference in male juvenile growth between the large‐ and small‐brained lines. Guppies have a pronounced sexual size dimorphism with males being much smaller than females Houde , and conThe small quantities of feces produced by guppies and the relatively large requirements of sample volume for these analyses mean that the estimates of DE are based on a large number of individuals, but relatively few measurements. This may decrease statistical power and it might therefore be premature to conclude that large‐ and small‐brained animals do not show subtle differences in DE. However, the demonstration of decelerated growth in the large‐brained females renders a prominent role of altered DE in the evolution of brain and gut size unlikely.In light of the many costs associated with increased encephalization, it stands clear that substantial benefits are required for larger brains to evolve. It has been suggested that such benefits mainly stem from increased ecologically relevant cognitive abilities associated with larger brains (e.g., Sol et al. To conclude, we do not find any differences in feeding propensity or DE between guppies selected for large or small brains. Instead, we find that large‐brained females have slower growth than small‐brained females during the first 10‐week period after birth. Together, our results suggest that there is no compensation in either feeding propensity or DE in the large‐brained lines, at least not within the current experimental set‐up. Interestingly, our study identifies reduced growth rate as an additional cost of evolving and maintaining a larger brain and thus we conclude that large brains can only evolve in situations where the benefits of larger brains are substantial. High predation may be a factor of particular relevance (Kotrschal et al. 10.5061/dryad.ns141.The doi for our data is doi:TableS1Supporting Click here for additional data file.


UPR) at the centre of pathological processes leading to neurodegenerative disease. The translational repression caused by UPR activation starves neurons of the essential proteins they need to function and survive. Restoration of protein synthesis, via genetic or pharmacological means, is neuroprotective in animal models, prolonging survival. This is of great interest due to the observation of UPR activation in the post mortem brains of patients with Alzheimer's, Parkinson's, tauopathies and prion diseases. Protein synthesis is also an essential step in the formation of new memories. Restoring translation in disease or increasing protein synthesis from basal levels has been shown to improve memory in numerous models. As neurodegenerative diseases often present with memory impairments, targeting the UPR to both provide neuroprotection and enhance memory provides an extremely exciting novel therapeutic target.Recent evidence has placed the unfolded protein response ( The development of disease‐modifying therapies for neurodegenerative diseases remains one of the biggest challenges facing society worldwide. As life expectancy increases, a concurrent rise in neurodegenerative diseases is occurring globally as the population ages. These disorders, which include Alzheimer's disease (AD), Parkinson's disease (PD), frontotemporal dementia (FTD) and tauopathies, as well as amyotrophic lateral sclerosis (ALS) and prion diseases, have distinct clinical, pathological and biochemical signatures, and all involve the accumulation of disease‐specific misfolded proteins in the brain. They are now collectively termed protein misfolding disorders post mortem brains. Amyloid plaques are mainly composed of amyloid‐β (Aβ) peptides, typically 1–40 and 1–42 amino acids in size, that are produced from the cleavage of the amyloid precursor protein (APP) by secretases AD is the most common cause of dementia and is characterized by episodic memory loss, progressive cognitive impairment and behavioural changes. Pathologically, amyloid plaques and neurofibrillary tangles are seen in C) by the misfolded form, termed PrPSc, via an auto‐catalytic post‐translational change in conformation. As neurons become depleted of PrPC, newly synthesized PrPC is produced, providing additional substrate for the conversion. PrPSc can spread between neurons, gradually increasing the area of the brain affected, or between organisms if infected material is ingested or transferred by iatrogenic exposure. It is now apparent that Aβ, tau and α‐synuclein are capable of the spreading templated conformational change first described for prions. It has been shown that this can cause spread between cells in vitro [It is increasingly clear that this group of diseases have common features as well as specific characteristics. Prion diseases are transmissible, and the mechanism of infectivity and spread involves conversion of native PrP  stress The UPR is a protective cellular response induced during periods of ER stress that aims to reduce unfolded protein load and restore protein‐folding homeostasis, ‘proteostasis’. Secreted and transmembrane proteins enter the ER as unfolded proteins to be properly assembled or to be targeted for degradation Paradoxically, the translation of some proteins is increased after eIF2α phosphorylation, due to the presence of multiple open reading frames in their 5′ untranslated regions The IRE1 and ATF6 arms of the UPR aim to increase the protein folding ability of the cell via the induction of chaperones and facilitate the removal of terminally misfolded proteins. Activation of IRE1 by dimerization and autophosphorylation enables its endoribonuclease activity and catalyses the splicing of x‐box binding protein 1 (XBP1) mRNA post mortem patients, the regions of the brain most affected by PSP EIF2AK3The UPR is central to the cell's response to dysregulated proteostasis. Markers of UPR activation including PERK‐P and eIF2α‐P have been reported in the brains of patients with AD, PD, ALS, the tauopathy progressive supranuclear palsy (PSP) and prion disease , 35, 36.+/− mouse model of prion disease that succumbs to Rocky Mountain Laboratory prion infection in around 12 weeks [There is an obvious correlation between UPR markers and neuropathology, but does UPR activation actively contribute to these diseases? Recently, we directly linked UPR activation to neurodegeneration in prion‐diseased mice. We used the Tg3712 weeks , 45, 46.We first tested the hypothesis that reducing eIF2α‐P would restore translation and prevent neurodegeneration  levels in transgenic mice as well as in AD patient brains et al. and Lourenco et al., discussed in more detail below, also provide evidence for a direct role of the UPR in AD [UPR activation is also associated with hyperphosphorylated tau. PERK‐P has been observed in neurons and glia that exhibit tau pathology, and is upregulated during the early phase of disease PR in AD , 67.In PD, mutant α‐synuclein has also been shown to accumulate within the ER, directly activating the PERK arm of the UPR by binding to BiP Approximately 2% of ALS patients have a mutation in the SOD1 gene and transgenic rodents expressing the mSOD1 are the most commonly used model of study in ALS research Although the UPR is normally a protective cellular response, the data presented above demonstrate that its dysregulation has a central role in many diseases. Chronic UPR activation, and the resulting swith from cytoprotective to cytotoxic signalling, contributes to disease. There are a number of unresolved questions, including: Why does the UPR become chronically activated and what causes the switch to cytotoxic signalling? If the majority of the disease‐modifying proteins discussed above do not build up in the ER lumen, why is the UPR activated? Is ER stress a response to neurodegeneration or does it contribute to disease initiation?These questions are now beginning to be answered. In most cases, it is likely that the induction of ER stress in neurodegenerative disease is not caused by misfolded protein build up in the ER lumen. However, some examples exist where disease‐associated misfolded proteins do build up in the ER. mSOD1 can translocate into the ER and has been observed bound to BiP and the foldase PDI Sc itself, is a cause of the ER stress in prion disease. Further investigations are also uncovering mechanisms of UPR activation caused by other misfolded proteins that do not build up in the ER. Abisambra et al. investigated how tau can activate the UPR using the tg4510 mouse model and an inducible tau cell model As discussed above, it is possible that the increased transcription of PrP, rather than the buildup of misfolded PrPde novo protein synthesis Protein synthesis is also central to memory formation, particularly via eIF2α modulation. This is important in this context, as memory loss is very often a central feature of neurodegenerative disease, especially in dementia. Neurophysiologically, memories are usually divided into short‐term memory, typically classed as lasting only a few hours in duration, and long‐term memory, lasting for years or even the lifespan of the organism, but there is no absolute distinction between memory types de novo protein synthesis. Significantly, ATF4 is a repressor of cAMP response element‐binding protein (CREB)‐mediated gene expression, which is essential for long‐lasting changes in synaptic plasticity and memory So how is the UPR involved in memory formation and consolidation? The translational attenuation of protein synthesis after eIF2α phosphorylation will prevent many of the proteins required from being produced during periods of ER stress that are required for LTP and long‐term memory formation. Although dendrites have a pool of mRNA and translational machinery at the synapse, this is not resistant to eIF2α‐P, and any longer term memory consolidation or reconsolidation will require Protein synthesis is therefore an essential part of memory storage, and the UPR tightly regulates protein synthesis rates, especially during periods of stress. This presents the question: can modulating the UPR enhance memory? Genetic reduction of eIF2α‐P in hippocampal slices from mice, either lacking GCN2 or heterozygous for a mutated eIF2α that cannot be phosphorylated, reduced the threshold for the induction of both LTP and learning in several behavioral tests , 87. Conet al. demonstrated that brain inflammation in AD models engages PKR to induce synaptic loss and memory impairments et al. also demonstrated that these deletions can protect against memory impairment and neurodegeneration in AD model mice Several recent studies have explored the link between UPR activation, memory and neurodegeneration. Lourenco It is unclear if the improvements in memory after UPR inhibition in these disease models are due to the release of translational repression that was preventing memory formation, or a consequence of the neuroprotection conferred. However, it is possible to improve memory using small molecules targeting downstream UPR signalling. An interesting study identified a small molecule, ISRIB, that inhibits downstream signalling of eIF2α‐P The UPR is the focus of increasing interest as an attractive target for disease‐modifying novel therapies due to its emerging role in the pathogenesis of neurodegenerative disease. The fact that these therapies are likely to increase memory in patients, where memory loss is a major problem, further improves the attractiveness of the UPR as a target. It remains to be seen if the problems associated with PERK inhibition can be abrogated. This, and other agents that mimic the action of ISRIB, for example, might hold the key to the first truly disease‐modifying and nonspecific treatment for neurodegeneration. Further work is needed to elucidate the exact nature of how these misfolded proteins cause ER stress and UPR activation, and the conditions that push UPR activation from a beneficial protective response to a central tenet of disease pathogenesis. UPR activation may also provide a useful biomarker for neurodegenerative disease.In conclusion, it is becoming increasingly clear that the translational repression due to phosphorylation of eIF2α is a key link between neuropathology and memory impairment in neurodegenerative disease. The reports discussed in this review suggest that restoration of protein synthesis through the eIF2α pathway is an attractive target to reduce memory impairment and prevent neurodegeneration across the spectrum of these disorders.


HIV viral load (VL) testing is an urgent public health priority. Implementation of testing is supported by the availability of accurate, nucleic acid based laboratory and point‐of‐care (POC) VL technologies and strong WHO guidance recommending routine testing to identify treatment failure. However, test implementation faces challenges related to the developing health systems in many low‐resource countries. The purpose of this commentary is to review the challenges and solutions from the large‐scale implementation of other diagnostic tests, namely nucleic‐acid based early infant HIV diagnosis (EID) and CD4 testing, and identify key lessons to inform the scale‐up of VL.The scale‐up of effective ART failure management and patient outcomes, reduce overall costs and help ensure the sustainable access to reduced pricing for test commodities, as well as improve supportive health systems such as efficient, and more rigorous quality assurance. These lessons draw from traditional laboratory practices as well as fields such as logistics, operations management and business.Experience with EID and CD4 testing provides many key lessons to inform VL implementation and may enable more effective and rapid scale‐up. The primary lessons from earlier implementation efforts are to strengthen linkage to clinical care after testing, and to improve the efficiency of testing. Opportunities to improve linkage include data systems to support the follow‐up of patients through the cascade of care and test delivery, rapid sample referral networks, and POC tests. Opportunities to increase testing efficiency include improvements to procurement and supply chain practices, well connected tiered laboratory networks with rational deployment of test capacity across different levels of health services, routine resource mapping and mobilization to ensure adequate resources for testing programs, and improved operational and quality management of testing services. If applied to VL testing programs, these approaches could help improve the impact of VL on EID and CD4 programs described here can be adapted to inform more effective scale‐up approaches for VL. They demonstrate that an integrated approach to health system strengthening focusing on key levers for test access such as data systems, supply efficiencies and network management. They also highlight the challenges with implementation and the need for more innovative approaches and effective partnerships to achieve equitable and cost‐effective test access.The lessons and innovations from large‐scale  In particular, the experience of CD4 assays and early infant HIV diagnosis (EID) testing over the past 15 years provide important lessons for VL implementation 2Over the past decade, public health programs in RLS have introduced and scaled up HIV early infant diagnosis and CD4 testing within many of the same laboratories and health facilities that are currently launching HIV VL testing. Testing coverage and volumes were initially modest – a few hundred thousand tests – but reached millions of tests within a few years It is expected that over 20 million people will need access to VL testing by 2020 3CD4 and EID implementation efforts have been heavily focused on building and equipping laboratories, training staff, and procuring test commodities. These activities were often highly successful, and both CD4 and EID laboratory capacity greatly exceeded test demand in many countries 3.1Long test turnaround times (TAT) are particularly devastating in the case of EID, where median TAT remain long, from 20 to 60 days Several countries have implemented improved sample referral networks and electronic result delivery to reduce TAT. This has included the mapping of sample referral routes and establishing disease‐integrated courier contracts to transport HIV, TB and other clinical specimens, e.g. with the Postal Service or Riders for Health. In Uganda, an extensive and robust sample referral network increased access to EID by 50% and reduced sample transport costs by 50% 3.2The use of point‐of‐care (POC) diagnostic technologies significantly improves retention in the pre‐ART care cascade. POC testing significantly reduced EID TAT and increased access to ART amongst HIV‐infected infants in Mozambique 3.3Other critical enablers of effective test utilization include integrated data and clinical systems that improve the cascade of care from the identification of eligible patients, the provision of testing, and delivery of results back to the health facility and patient. This enables results to be used swiftly to make clinical decisions. It is therefore important to build diagnostic systems with patient care in mind, ensuring that delivery of results to health facilities is well integrated with clinical patient management and well synchronized with the patient pathway of care. Several interventions have been shown to improve the cascade of clinical care from testing through linkage to care, including clinical quality improvements such as the use of SMS result delivery to clinics or patients, and active patient follow‐up by community health workers 3.4It will be useful to draw from the above interventions that have proven to be successful with CD4 and EID programs so that steps can be taken early in VL implementation to establish effective practices. In particular, testing programs need to ensure that VL testing is well integrated with ART failure management processes, for example that test TAT do not introduce long delays in detecting and confirming failure, and that results are delivered to clinicians and patients in order for timely follow‐on care, such as enhanced adherence support and/or a drug regimen switch. VL monitoring and evaluation programs should also include metrics such as testing coverage, test TAT, test delivery to clinics and patients, and test utilization to guide ART management. In many settings, VL programs can leverage existing EID and CD4 sample referral and electronic results delivery networks. The utility of POC VL still needs further investigation, but it is possible that the availability of rapid VL results may improve patient adherence, differentiated care, and reduce program costs Table .4The establishment of large scale CD4 and EID testing programs in low‐resource settings over the past 15 years faced many challenges 4.1Large‐scale VL testing programs will consume significant quantities of diagnostic commodities run on networks of instruments. Ensuring the reliable and efficient supply of testing products and preventing breakdowns of instruments requires some of the innovative approaches that have been developed within EID and CD4 testing programs. At the outset of these programs, test kit and instrument costs were high, test procurement was highly disaggregated across government, donors and implementing partners, and stock outs and instrument breakdowns were frequent. Many of these challenges have not yet been resolved; a recent WHO survey found approximately 10% of instruments were not in use due to stock‐outs, breakdowns or non‐installation Global access price deals are negotiated centrally and provide equitable access to low pricing for all public sector buyers irrespective of procurement volume. This allows smaller countries with limited purchasing power to access the same pricing as high volume countries. Price reductions of 30% to 50% for CD4 and EID tests have resulted in substantial commodity budget savings for countries and donors around the world, and have helped expand access to these tests Supply chain initiatives can have significant impact, reducing costs and increasing health product availability The Global Fund has established framework agreements with major VL suppliers that provide mechanisms for standardized access to bundled supply terms Although they are valuable tools for optimizing supply chain, bundled agreements have been infrequently used for VL testing programs, partly due to unconsolidated procurement or difficulty with undertaking multi‐year procurement commitments. As VL testing programs scale‐up to meet growing demand, the need for more effective procurement mechanisms and better coordinated procurement across buyers will also grow. Given the relatively high reagent and capital costs, and the technical complexity of VL instruments, and hence need for routine service by an engineer, carefully negotiated bundled agreements and access pricing will likely be useful measures to increase the sustainability and reliability of large‐scale testing programs.4.2Health systems in many LRS often have limited ability to support large testing networks The rational deployment of testing technologies within the networks is a key factor for efficiency. A common feature of CD4 and EID testing networks to date has been over‐capacity and under‐utilization of the instrument base. A 2013 WHO survey in 127 countries identified a surplus of global CD4 capacity, sufficient instrumentation to conduct 4.6 tests per year for every person living with HIV, and 12.8 tests/year for every person on ART, as opposed to the 1 to 2 tests per year required at that time 4.3One of the key lessons from the scale‐up of CD4 and EID programs is while significant donor funding has gone into laboratory services over the past decade, long‐term national investment in laboratory testing is needed. Effective national laboratory systems such as logistic and health data systems are often under‐funded and not fully part of routine service. This is partly because the full costs of testing services, especially when new diagnostics are being rapidly scaled up, and the financial resources needed to ensure access to essential diagnostic tests at national scale are difficult to estimate and track. As a result, large‐scale public testing programs such as VL face risks and may be unstable and unsustainable unless appropriate investments are made in testing and necessary supportive systems. For example, in Zimbabwe despite a goal of 21% VL coverage in 2015, only 5.6% test coverage was achieved due to challenges in resource mobilization and related factors In addition, the investment case for laboratory systems strengthening needs to be more clearly articulated so that sustainable funding for these systems can be established. Investments in scaling up CD4 and EID in many countries have often included laboratory system strengthening programs for logistics, data and quality, providing an opportunity to build on these investments to support VL testing programs 4.4The unprecedented scale of CD4 and EID testing programs has highlighted the need for stronger management within public laboratory programs, both operational and quality management. Laboratory quality improvement programs such as the Strengthening Laboratory Management Towards Accreditation (SLMTA), developed by the United States Centers for Disease Control and Prevention, and the WHO Strengthening Laboratory Improvement Towards Accreditation (SLIPTA) program supported by the African Society for Laboratory Medicine have increased quality management skills and have established a growing trend towards accreditation in public laboratories Tiered networks of laboratories have significant operational management needs to ensure efficiency; however most leaders of public health laboratory networks are not trained in operations 5The global scale‐up of EID and CD4 testing services has established important systems and experience with large‐scale public health testing programs. It has also identified weaknesses in diagnostic access that may ultimately limit the success of these programs, as well as new tests such as VL. There are many innovations and best practices from these initial large‐scale testing programs to learn from, as well as persistent challenges to overcome. There are critical needs and opportunities in linkage to care and testing efficiency, including improved data and sample referral systems, POC tests, improved supply terms, and more effective laboratory network management that are important to address during the scale‐up of HIV VL testing programs in resource limited settings Table . Learnin6The authors have no conflicts of interest.7TP, CZ, ZK, AE, BA, LV, AC, ND and IJ developed and finalized the manuscript.9Unitaid, PEPFAR.


Aging Cell, Evangelou and colleagues developed a sensitive method for identification of senescent cells in different types of biological material based on the detection of lipofuscin using an analogue of Sudan Black B (SBB) histochemical dye coupled with biotin, which they named GL13. The authors propose that this method is more sensitive and versatile than using SBB alone. Lipofuscin, a nondegradable oxidation product of lipids, proteins and metals, is found in senescent cells. Detection of lipofuscin using GL13 staining may be a more feasible method than others currently used for identification of senescent cells both in cell culture and tissues.Cellular senescence is a state of irreversible cell cycle arrest induced by different types of cellular stresses. The field of senescence has made significant advances in the understanding of many of the mechanisms governing this phenomenon; however, a universal biomarker that unambiguously distinguishes senescent from proliferating cells has not been found. In this issue of  Cellular senescence has been defined as an irreversible cell cycle arrest which stops the propagation of damaged cells. It was first observed by Hayflick and Moorhead who demonstrated a limited replicative lifespan of human fibroblasts in culture  activity at pH 6 is probably the most widely utilized method for identification of senescent cells (Dimri et al., et al., et al., et al., et al., Examples of currently used markers are as follows: increased expression of cyclin kinase inhibitors p21 and p16 and absence of proliferation markers; telomere‐associated DNA damage foci (Hewitt et al., Lipofuscin is a nondegradable aggregate of oxidized lipids, covalently cross‐linked proteins, oligosaccharides and transition metals which accumulate within lysosomes. It is a product of iron‐catalysed oxidation and polymerization reactions of a variety of cellular structures and macromolecules (Terman & Brunk, In this article, Evangekou and colleagues designed and synthesized a structurally similar compound to SBB and coupled it to biotin. Commercially available SBB contain numerous impurities which impact on staining quality and justified the need to synthesize a new analogue. The chemical coupling with biotin allows its detection using antibiotin antibodies and thereby increases its detection sensitivity Fig. . The autThe authors present multiple examples of positively stained cultured cells induced to senescence by replicative exhaustion, genotoxic stress, overexpression of p21 or oncogene activation. Furthermore, they reveal that mouse models of cellular senescence and human clinical samples of malignant lesions and irradiated tissue are characterized by elevated numbers of GL13‐positive cells, when compared to controls.Despite its obvious advantages, users should be aware of some possible limitations of the method. For instance, lysosomes may be loaded with degradable material, described as ‘temporary lipofuscin’, but also lipid droplets and glycogen which may be detected by methods such as SBB (Terman & Brunk, et al., et al., In addition, age‐dependent accumulation of lipofuscin has been shown to occur in postmitotic tissues which in theory should not experience the phenotype originally described by Hayflick. This is at odds with the idea that lipofuscin accumulation is specific to senescent cells. One explanation is that similar pathways which drive senescence in mitotic cells can also be activated in postmitotic cells, as demonstrated in neurons (Jurk in vivo. Finally, strategies such as the recently developed mouse models where p16‐expressing senescent cells can be specifically eliminated (Demaria et al., et al., While the authors have convincingly demonstrated that lipofuscin accumulation correlates with senescent markers in cell culture and that lipofuscin increases in tissues with age, future work should investigate more thoroughly whether and to what extent the lipofuscin signal overlaps with other established senescent markers such as SA‐β‐Gal or p16 expression during aging et al., A separate question which arises from this work is whether lipofuscin accumulation is a mere consequence of the induction of the senescence programme or whether its accumulation contributes causally to the development of senescence. The latter hypothesis would be consistent with early findings which demonstrated that synthetic lipofuscin could induce senescence in human fibroblasts (von Zglinicki in vivo. However, one major challenge for the community is the identification of specific markers. The novel method proposed by Evangelou and colleagues offers a possible easy and versatile solution, but whether it works as a standalone marker or should be used in combination with several others remains to be determined.The last decade has seen incredible advances in the field of cellular senescence with the realization that senescent cells play important, albeit distinct, roles Work in JP's laboratory is funded by the Biotechnology and Biological Sciences Research Council BBSRC.The authors declare no conflict of interests.


To examine older adults’ perspectives regarding managing sleep problems through selection and use of non‐prescription sleep aids, and the role of pharmacists.Telephone interviews were conducted from May to June 2015 with 116 individuals aged ≥60 years in Pittsburgh, Pennsylvania. Participants reported in a previous survey to have used at least one non‐prescription sleep aid in the past 30 days and were willing to participate in a follow‐up interview. Interview guides were designed to elicit perspectives of sleep problems, selection and use of non‐prescription sleep aids, and consultation with healthcare professionals. Interview transcripts underwent content analysis.Four themes emerged as follows: experiences with sleep problems, selection of non‐prescription sleep aids, non‐prescription sleep aid use and interactions with healthcare professionals. Over half of participants reported using a non‐prescription sleep aid for >1 year, were satisfied with its use and perceived it improved sleep quality. Participants commonly used an antihistamine‐only sleep aid; 36% of participants self‐recommended their sleep aid; and 16% of participants consulted healthcare professionals. Few participants read medication dosage labels (22%), side effects or warnings (19%), and many reported they disregarded directions. Participants did not typically consult pharmacists about sleep problems (65%) but perceived that they could assist with medication concerns.Although most participants had favourable perceptions of non‐prescription sleep aids, older adults may be inappropriately using non‐prescription sleep aids to self‐manage sleep problems by frequently disregarding medication labels and directions for safe use. Also, few older adults are discussing their sleep aid selection and use with pharmacists. Approximately 6.3 million older adults in the United States report having sleep problems,Many factors potentially impact proper sleep hygiene including napping during the day, inconsistent bedtime, consumption of stimulants or food too close to bedtime, insufficient exercise and reading or watching TV in bed.A recent systematic review found a lack of robust clinical evidence for the safety and efficacy of common non‐prescription sleep aids, including first‐line antihistamines such as diphenhydramine or doxylamine and supplements such as melatonin and valerian, to treat occasional disturbed sleep or transient insomnia.To our knowledge, little is known about older adults’ perspectives regarding management of sleep problems, their decision‐making process to select and use non‐prescription sleep aids and consultation with healthcare professionals on appropriate selection. Further research could elucidate these decision‐making processes and targeted interventions to improve older adult patient safety by healthcare professionals such as geriatricians and community pharmacists. Through consultation, healthcare professionals may be able to identify specific sleep hygiene factors that could be influencing sleep problems and suggest non‐pharmacological  interventions to improve sleep. Healthcare professionals, such as community pharmacists, can educate older adult patients taking OTC sleep aids on the potential risks and assist them in selecting appropriate products to improve patient safety. It has been recommended that pharmacists have a more significant role in counselling older adults on appropriate use of non‐prescription sleep aids;The study sample was a subset of individuals that participated in a previous mail survey sent in February 2015 to 2064 members of the Pittsburgh Claude D. Pepper Older Americans Independence Center. The Pepper Community Registry includes over 2000 community‐dwelling persons in the Pittsburgh, Pennsylvania region, aged 60 and older who consented to be contacted for participation in Pepper‐approved research studies. The distributed survey was designed to elicit information on older adults’ perspectives of sleep quality and satisfaction, along with methods of alleviating sleep problems such as the use of OTC medications.Semistructured telephone interviews were utilized in data collection, and participants’ verbal consent was obtained. An interview guide , melatonin (30%) and combination antihistamine/analgesics (29%). Among the 36 antihistamine‐only products being used, 27 contained only diphenhydramine and nine contained only doxylamine. Among the 34 antihistamine–analgesic combination products reported, 22 were acetaminophen/diphenhydramine, 10 were ibuprofen/diphenhydramine, one was naproxen/diphenhydramine, and one was acetaminophen/chlorpheniramine.Four major themes emerged including (1) experiences with sleep problems, 2) selection of non‐prescription sleep aids, (3) non‐prescription sleep aid use and (4) interactions with healthcare professionals. Themes and related verbatim quotes from participants are presented in Table  selectioParticipants stated a variety of causes they considered to be associated with their sleep problems. Participant‐reported causes of sleep problems included worrying and stress (28%), caffeine use (22%), frequent night‐time bathroom use (20%) and other non‐sleep‐related ailments, like comorbidities (16%) and pain (16%). However, older adults most commonly reported being unsure of the cause of their sleep problems (29%).Approximately one in five participants stated that their sleep problems had lasted over 10 years. Most participants reported that their sleep problems did not affect their mood (65%) or daily life (54%), but some participants said that their mood was negatively impacted or they were chronically tired from their sleep problems . A majority of participants assumed their sleep problem did not require medical attention. Although participants used non‐prescription sleep aids, 23% also used reading as a non‐pharmacological approach to improve sleep.More than twice as many participants chose their sleep aid based on self‐recommendation rather than by consulting with a healthcare professional (36% versus 16%). Just over one in five participants compared different non‐prescription sleep aids during the decision‐making process (22%). Many participants did not mention a specific rationale for selection of a particular sleep aid, and very few participants had consulted with a healthcare professional about selection. Exactly half of the participants reported reading the sleep aid label at least once before making a selection. Familiarity through branding or media was also a factor in some participants’ choice of non‐prescription sleep aids.Forty percent of participants reported daily or frequent use of a non‐prescription sleep aid, and 56% reported using a non‐prescription sleep aid for more than one year. Few participants reported reading the non‐prescription sleep aid label regarding dosage information (22%) or side effects and warnings (19%), and many reported they had ignored or disregarded directions. Some participants mentioned the need for the labels to have larger print (17%). Nearly half of participants reported consuming alcohol on the same nights they used their sleep aids (45%). Participants were typically satisfied with their non‐prescription sleep aid (54%) and perceived improved quality of sleep with use (53%). Some participants reported being neither satisfied nor dissatisfied with their non‐prescription sleep aid (20%) and were unsure of the resulting quality of sleep (16%). Most participants reported they did not experience any side effects after taking the non‐prescription sleep aid; however, those that stated they did typically had drowsiness the following morning and experienced difficulty in waking up.While the majority of participants reported they had discussed their sleep problems with a healthcare professional (66%), far fewer had received consultation about non‐prescription sleep aid use (22%). Nearly one‐third of participants reported that their healthcare professionals were not aware of their non‐prescription sleep aid use (29%), and almost half (47%) had not discussed the safe use and side effects of non‐prescription sleep aids with their physician or pharmacist. The majority of participants reported they did not consult a pharmacist about their sleep problems (65%). One‐third of participants reported that they preferred provision of counselling related to sleep health from a physician rather than a pharmacist. A little over 20% of participants perceived that pharmacists only address medication concerns and not other aspects of health. About 24% of participants reported being comfortable with pharmacists, and 16% perceived pharmacists as being educated. Most participants felt that privacy was not a concern when discussing sleep problems and treatment with a community pharmacist.Findings indicate that older adults encounter sleep problems due to various causes, and may often be self‐treating with non‐prescription sleep aids without awareness of safety risks or consultation with a healthcare professional.The strengths of this study include the use of semistructured interviews that allowed for further discussion and clarification of questions with participants. Study findings contribute valuable evidence to the literature regarding older adults’ perspectives about their use of non‐prescription sleep aids. This study also has recognized limitations. Results may not be generalizable, because the study sample was drawn from a research registry of older adults living only in the Pittsburgh, Pennsylvania region, in the United States. Participants were predominantly female, White and well educated and thus are not representative of all older adults. Our qualitative study did not allow for determination of associations between participant characteristics and satisfaction with sleep aid use. We did not examine patient history or concurrent medication use, which may affect older adults’ use of non‐prescription sleep aids. Additionally, the usefulness of the information given by healthcare professionals was not evaluated.Participants reported causes of difficulty falling or staying asleep which included anxiety, caffeine intake, bathroom use, comorbidities and pain, although some participants did not know the cause of their sleep problems. Older adults are typically more medically complex,Participants reported the use of pain relievers/fever reducers without diphenhydramine to improve their sleep. The use of such products may suggest that pain is negatively impacting sleep in older adults, and they are self‐treating their pain using OTC medications. This was further supported by our study findings, as 16% of participants reported pain and 16% reported non‐sleep‐related ailments such as comorbidities to be causes of their sleep problems. Determining the possible contribution of comorbidities and pain to diminished sleep quality may likely be important in addressing older adults’ sleep problems and identifying appropriate treatments.Our study showed that more participants were selecting non‐prescription sleep aids based on self‐recommendation rather than by consulting a healthcare professional, although community pharmacists might be present at purchase. Some participants noted being familiar with brands of certain non‐prescription sleep aids through advertising or media which significantly influenced their decision‐making process and selection. Knowledge of a non‐prescription sleep aid may be based on advertisements that could be misleadingStudy participants were typically satisfied with their sleep aids and perceived improved quality of sleep with use. Participants primarily reported taking non‐prescription sleep aids containing diphenhydramine and doxylamine, which the Beers Criteria for Potentially Inappropriate Medication Use in Older Adults strongly recommends avoidance of due to possible harmful effects.Many unsafe behaviours related to non‐prescription sleep aid use were reported. Although non‐prescription sleep aids are not meant to treat chronic sleep problems, nearly half of participants used them daily or very often, and more than half reported use for more than a year. Few participants reported consulting the label for dosage information, and even fewer for side effects and warnings. This could possibly result in older adults taking non‐prescription sleep aids at a higher than recommended dose, for a longer duration, and without awareness of potentially dangerous side effects or interactions.Nearly 40% of older adults in the United States are taking five or more prescription medications.Although alcohol can exacerbate the sedative effects of non‐prescription sleep aids, nearly half of participants reported consuming alcohol on the same nights as sleep aids. Many sleep medications have been contraindicated with alcohol use due to unsafe interactions.Medication labels on non‐prescription sleep aids containing diphenhydramine or doxylamine warn that patients should consult a doctor if they need to use the sleep aid for longer than two weeks and alcohol should not be consumed with use. However, the text size is often small and some study participants reported experiencing trouble reading such information. Older adults with lower levels of health literacy may also have more difficulty understanding medication label information. Larger and clearer font Older adults could receive consultation about their sleep hygiene and safe non‐prescription medication use from healthcare professionals such as doctors, nurses or pharmacists. Community pharmacists are medication experts that are easily accessible where non‐prescription sleep aids are sold; however, few of the participants in this study had used them as a resource for discussing their sleep problems and receiving information about appropriate OTC medication use. This may be due in part to the belief that community pharmacists primarily address questions about prescription medications and not health concerns. Often, participants reported they typically would discuss sleep problems with their physicians and did not think of asking pharmacists about health or medication concerns. However, some participants stated that they would be comfortable discussing health concerns with pharmacists and considered them to be well educated. Overcoming these perceptions of community pharmacists could potentially encourage older adults to seek medication counselling from these healthcare professionals when selecting and purchasing non‐prescription sleep aids in pharmacies to improve patient safety.Findings indicated that many older adults experience sleep problems due to various causes, often from worrying, stress or caffeine use. Many older adults self‐treat sleep problems with non‐prescription sleep aids and are often taking potentially inappropriate products containing diphenhydramine or doxylamine, and many are doing so for longer than these products are intended to be used. Over half of participants reported using a non‐prescription sleep aid for more than one year, were satisfied with its use and perceived it improved sleep quality. The decision‐making processes in choosing non‐prescription sleep aids do not appear to involve appropriate attention to dangerous risks and side effects on the medication label, with frequent disregard to directions and minimal healthcare professional consultation. Community pharmacists are easily accessible and able to provide medication counselling related to non‐prescription sleep aid use, but patients tend to perceive them as medication dispensers, rather than resources for addressing health or medication‐related concerns. Future investigations should focus on development of patient‐centred interventions to facilitate patient–pharmacist engagement in community pharmacies to support older adults’ safe and appropriate treatment of sleep problems, while minimizing the risks of potentially inappropriate non‐prescription sleep aids containing diphenhydramine or doxylamine.The Author(s) declare(s) that they have no conflicts of interest to disclose.This work was supported by the Academy Health New Investigator Small Grant Program (NISGP).All authors contributed to (1) study design; (2) collection, analysis and interpretation of data; (3) the writing of this report; and (4) the decision to submit the manuscript for publication. All Authors state that they had complete access to the study data that support the publication.Appendix S1. Older Adult Interview Guide.Click here for additional data file.


Hum Brain Mapp 37:54–66, 2016. © 2015 The Authors Human Brain Mapping Published by Wiley Periodicals, Inc.Successful social interactions depend on the ability to anticipate other people's actions. Current conceptualizations of brain function propose that causes of sensory input are inferred through their integration with internal predictions generated in the observer's motor system during action observation. Less is known concerning how action prediction changes with age. Previously we showed that internal action representations are less specific in older compared with younger adults at behavioral and neural levels. Here, we characterize how neural activity varies while healthy older adults aged 56–71 years predict the time‐course of an unfolding action as well as the relation to task performance. By using fMRI, brain activity was measured while participants observed partly occluded actions and judged the temporal coherence of the action continuation that was manipulated. We found that neural activity in frontoparietal and occipitotemporal regions increased the more an action continuation was shifted backwards in time. Action continuations that were shifted towards the future preferentially engaged early visual cortices. Increasing age was associated with neural activity that extended from posterior to anterior regions in frontal and superior temporal cortices. Lower sensitivity in action prediction resulted in activity increases in the caudate. These results imply that the neural implementation of predicting actions undergoes similar changes as the neural process of executing actions in older adults. The comparison between internal predictions and sensory input seems to become less precise with age leading to difficulties in anticipating observed actions accurately, possibly due to less specific internal action models.  From skilled movements, like a penalty kick in football, to everyday actions, like shaking hands, anticipating what other people will do next is fundamental for successful social interactions. According to the predictive coding framework, incoming sensory signals are matched with internally generated predictions that reflect an observer's model of the world to infer the causes of sensory input Clark, . During Less is known about functional changes in this network as the brain ages. Generally, older compared to younger adults show less selectivity in relevant neural networks and recruit additional regions during performance of a variety of tasks Grady, . It follAt the behavioral level, we previously found that older compared to younger adults predict the time‐course of observed actions less precisely, although task performance in both age groups benefited from sensorimotor experience in the observer [Diersch et al., en activity within this network should be greater the earlier in time the actions continue after occlusion  [cf., Cross et al., In line with previous research, we expected the prediction of partly occluded actions to engage brain regions typically involved in predictive processing of others' actions, such as frontoparietal and occipitotemporal cortices of the AON [cf., Bubic et al., M = 2.29 ± 0.65 vs. M = 1.65 ± 0.65) on a 5‐point rating scale ranging from “very good” to “very bad.” Their ratings about their past performance ability were more positive than those about their current ability, t(20) = 6.04, P < 0.001.After being screened for health status and potential contraindications to MRI scanning, 24 older adults were invited to take part in the fMRI experiment. Exclusion criteria included a history of any major physical or neurological disease, psychiatric disorder, and use of medication that might affect cognitive performance or blood flow. Participants provided written informed consent and were paid for their participation in accordance with the local ethics committee. Three participants were excluded from data analyses due to problems with following task instructions during scanning. The final sample consisted of 21 healthy older adults  with normal or corrected‐to‐normal vision. They were right‐handed (LQ = 92.9 ± 8.32); [Oldfield, During fMRI scanning, participants observed different action sequences consisting of simple movement exercises  that were performed by a female and male non‐athlete in a sports hall. The duration of the videos was 9.0 s on average (range: 8.0–10.9 s). A prediction trial started with a fixation cross (1000 ms), followed by the beginning of an action sequence. The actions were visible for 4.5 s on average (range: 3.1–5.9 s) before they were occluded for 1000 ms by a grey rectangle. The continuation after the occlusion was either congruent, temporally too early or too late on two different levels  during which the temporal coherence of the four incongruent continuations  had to be judged. Participants received feedback of their performance. Because participants were always forced to decide whether the actions continued too early or too late, the congruent continuation was not presented at this stage due to a lack of a correct response option. Time between the training and fMRI scanning was 43.3 ± 28.5 days on average.3.Scanning was performed on a 3T Philips Achieva scanner with a SENSE 8‐channel phased‐array head coil. In two functional runs, 459 whole‐brain EPI images were acquired in ascending order  with a repetition time of 2000 ms . Imaging parameters were as follows: echo time, 30 ms; slice thickness, 4mm; no interslice gap; in‐plane resolution, 3 mm × 3 mm; acquisition matrix, 76 × 75 voxels; and flip angle, 90°. High‐resolution anatomical images were acquired using a T1‐weighted 3D magnetization‐prepared rapid gradient echo (MPRAGE) sequence with the following parameters: repetition time, 12 ms; echo time, 3.5 ms; flip angle, 8°; image matrix, 240 × 240 voxels; spatial resolution, 2 × 2 × 2 mmIn addition to a standard analysis of variance (ANOVA) on the proportion of too early responses with continuation after occlusion as repeated‐measures variable, the timing and sensitivity in action prediction was analyzed psychophysically. By fitting a psychometric function over the z‐transformed too early response rates on every continuation after occlusion by means of a linear regression, two psychophysical indices could be derived: (1) the point of subjective equality (PSE), which is the point in time at which participants judged the action continuation on chance level, that is, at which they perceived it as being just‐in‐time  and (2) the just‐noticeable difference (JND), which represents the difference that is reliably noticed in half of the cases (75% − 25%)/2 or the steepness of the psychometric function  [cf., Gescheider, t‐tests. We further checked for areas where activity changed as a function of the temporal shift from the earliest to the latest continuations and vice versa by using two linear contrasts  over continuations after occlusion (−800 to +800 ms). Changes in neural activity as a function of temporal distance to the congruent continuation were examined by means of two additional polynomial contrasts  over continuations after occlusion. This allowed us to examine the extent to which AON engagement is modulated by the temporal shift of the continuation after occlusion, either depending on whether the action continuation is shifted backwards or forwards in time or depending on the extent of deviation from the correct continuation after occlusion.Image preprocessing and statistical analyses were performed in SPM8 . Preprocessing of the EPI volumes included realignment, slice timing, and spatial normalization to MNI space [Ashburner and Friston, In a second GLM, correct and incorrect predictions on incongruent continuations after occlusion were separately modeled . This allowed us to test whether erroneous predictions might be associated with different neural activation patterns than correct predictions on too early and too late continuations after occlusion. Each of these regressors contained both levels of temporal delay (±400/±800 ms) to ensure that a reasonable number of valid events enter the analysis. All the other regressors were modeled in the same way as in the first GLM.In additional analyses, the relation between neural responses and between‐subject variability was examined in more detail. Therefore, the individual contrast images for the comparison between prediction and baseline from the first GLM were entered into a multiple regression analysis. Values for the timing in action prediction , the sensitivity in action prediction  that were based on a psychometric function fitted across responses on the five continuations after occlusion, and, given the large age range in our sample, the age of the observer served as predictor variables. This allowed us to identify the unique contribution of each source onto neural activation patterns while ensuring that both the neural measure  and the behavioral indices share the same basis, that is, responses across every continuation after occlusion. The sex of the observer was additionally included as covariate of no interest due to a correlation between the JND and sex observed at the behavioral level.http://surfer.nmr.mgh.harvard.edu) [Dale et al., To take into account our a priori hypotheses about the relation between neural activity and between‐subject variability, anatomical regions of interest (ROI) were defined for regions consistently implicated in action observation (the AON) Grafton,  and founP < 0.001 (uncorrected). Whole brain analyses focus on brain regions reaching a cluster‐corrected threshold of P < 0.05, family wise‐error (FWE) corrected. For the analyses of the effects of age and prediction performance on neural activity, we applied small volume corrections for the brain volume determined by the respective ROI . Anatomical localizations of significantly activated clusters were aided by the Anatomy Toolbox in SPM8 [Eickhoff et al., All analyses were conducted with a voxel‐level height threshold of F = 195, P < 0.001. The proportion of too early responses declined linearly across time as indicated by a significant linear trend model, F = 924, P < 0.001  = 1.04, P = .312, whereas for every incongruent continuation participants performed well above chance level, all t ≥ 4.63, P ≤ 0.001. Performance of the sample was not biased towards the past or the future as implied by a PSE of −29.3 ± 270 ms that did not differ significantly from zero, t(20) = 0.50, P = 0.625. However, a relatively large time‐bin around the congruent continuation was predominantly perceived as being just‐in‐time (JND = 440 ± 105 ms).An ANOVA on the proportion of too early responses with continuation after occlusion  as repeated‐measures variable revealed a significant main effect of continuation after occlusion, del, F1,0 = 924, r ≤ .290, P ≥ 0.201, two‐tailed). This confirms that experience with the observed actions did not influence performance on the task, as all participants were highly familiar with the observed actions. Previously, we showed that the JND but not the PSE is affected by the age of the observer when comparing older and younger adults [Diersch et al., rho = −0.553, P = 0.009, two‐tailed) implying that female observers of the sample showed a higher uncertainty about the time‐course of the action continuations than male observers. This might be attributable to a sampling bias in the present study since we did not observe similar effects in our previous studies [Diersch et al., The prediction timing and sensitivity were not related to the motor familiarity or age of the observer, as suggested by nonsignificant correlations between PSE or JND values and the age of the observer or experience with observed actions  and pre‐supplementary motor area (pre‐SMA), in the bilateral anterior insula extending to the basal ganglia with peaks in the putamen, the thalamus, and the brainstem Fig. . No diffNext, we examined in which regions activity changed linearly as a function of the temporal shift of the continuation after occlusion by means of two linear contrasts. This analysis showed that activity in the right IFG and IPL together with the bilateral posterior temporal gyrus at the transition to the lateral occipital cortex  increased the earlier in time the actions continued after occlusion Fig. . The invWhen comparing neural activity on incongruent (too early and too late) continuations after occlusion while taking only correctly predicted action sequences into account, a similar pattern was obtained as in the linear contrast analyses in the first GLM. For the comparison between correctly predicted too early continuations and correctly predicted too late continuations, enhanced activity in right IFG and IPL as well as in bilateral occipitotemporal cortices was found Table . Supra‐tP ≤ 0.041) and approaching significance when correcting for multiple comparisons at the cluster‐level , the sensitivity in action prediction , and age as predictor variables while controlling for the sex of the observer. We additionally explored whether individual differences on these measures were associated with activity changes in regions elsewhere in the brain. Increasing age in our sample (aged 56–71 years) was associated with increasing activity in the anterior portion of the right IFG ROI that belonged to a larger cluster in the right middle frontal gyrus/frontopolar cortex when considering the entire brain Fig. . The ROIP = 0.049) and was marginally significant when correcting for multiple comparisons at the voxel‐level (P = 0.064). There was no region in which activity correlated negatively with the JND. We did not observe any significant effects of the timing in action prediction  within the ROIs or elsewhere in the brain.We further examined whether a lower sensitivity in action prediction  was associated with greater engagement of the caudate nucleus by means of a ROI analysis. In line with our hypothesis, we found that the amplitude of activity in the left head of the caudate ROI correlated positively with the JND of the observer Fig. , Table 3The prediction of observed actions is less precise and is accompanied with more extended brain activations in older compared to younger adults [Diersch et al., Crucially, neural activity was modulated by the point in time to which the action continuation after occlusion referred: the more the action continuation was shifted backwards in time, reflecting action states that happened during the occlusion period, the greater parts of the AON were engaged. Conversely, early visual cortices were more engaged the more the action continuation after occlusion was shifted towards the future. This pattern was also highly evident when incorrectly predicted action continuations were excluded from the analysis. This suggests that older adults, like younger adults, generate internal predictions about the most‐likely action trajectory with the help of their sensorimotor system during the occlusion period [cf., Cross et al., This is further supported by the observed differences in neural activity between correct and erroneous predictions that varied as a function of the temporal shift of the continuation after occlusion. We found that the pre‐SMA is generally more engaged when the actions continued too early after occlusion, irrespective of participants' judgements, compared with correctly predicted too late continuations. Higher activity in the pre‐SMA was also found when comparing incorrectly predicted too late continuations to correctly predicted too late continuations. In younger adults, the pre‐SMA has been associated with action monitoring and ordering movements in time [Nachev et al., Importantly, the time of occlusion as well as the amount of visual input after occlusion was the same between too early, congruent, and too late continuations. Differences in neural activation patterns therefore cannot be ascribed to differences in the amount of action information being presented. Moreover, brain activity did not vary as a function of the temporal deviation of the observed continuation from the congruent continuation. Given that each continuation after occlusion appeared equally often and participants were trained on the task beforehand, they expected the action continuations to be shifted in time. Thus, neural activity measured at the beginning of occlusion did not seem to reflect the mismatch between the internal prediction and sensory input  but rather the neural implementation of action prediction.Participants in our sample ranged in age from 56 to 71. We identified two areas in the brain where neural activity was positively correlated with the age of the observer, both of them in close vicinity to classical AON sites in the younger brain. Specifically, activity increases were found in anterior IFG that extended to the middle frontal gyrus/frontopolar cortex as well as in anterior STG/STS. This implies that neural activity becomes less specific and spreads from posterior IFG and STS to more anterior parts of these regions in older adults, which already becomes evident when considering an age range of 15 years beginning at about the age of 56. In younger adults, the anterior IFG has been implicated in encoding more abstract features of observed actions, such as their goal and intention [Press et al., Interestingly, age‐related activity increases in regions such as the PFC are a common finding in the motor control literature [Seidler et al., The only region that showed performance‐dependent activity changes in our sample was the caudate. The lower the sensitivity in action prediction, the higher was the BOLD response in this region. Caligiore et al.  argued tHere, we show for the first time that neural activity in the AON of healthy older adults during the prediction of partly occluded actions is sensitive to the time point to which the action continuations after occlusion refer. We further found that neural activity becomes less distinct in parts of the AON between 56 and 71 years of age, whereas lower prediction sensitivity is linked to increases in striatal activity. Similar changes have been observed in studies examining the neural processing of executed actions in older adults. In line with current models of age‐related changes in brain function based on the predictive coding framework, the results suggest that ability to accurately predict actions performed by others declines with advancing age as a result of less specific internal action models and difficulties in minimizing prediction error signals in response to sensory input.


Rational and generalisable methods for engineering surface functionality will be crucial to realising the technological potential of nanomaterials. Nanoparticle‐bound dynamic covalent exchange combines the error‐correcting and environment‐responsive features of equilibrium processes with the stability, structural precision, and vast diversity of covalent chemistry, defining a new and powerful approach for manipulating structure, function and properties at nanomaterial surfaces. Dynamic covalent nanoparticle (DCNP) building blocks thus present a whole host of possibilities for constructing adaptive systems, devices and materials that incorporate both nanoscale and molecular functional components. At the same time, DCNPs have the potential to reveal fundamental insights regarding dynamic and complex chemical systems confined to nanoscale interfaces. Tremendous synthetic and analytical advances over more than two decades have opened up a new region of chemical space on the nanoscale, leading to excitement on account of the extraordinary properties observed for myriad types of nanoparticles (NPs) and other materials in this size regime.The chemical nature of nanoscale surfaces is inherently critical to the behaviour of all nanomaterials. The canonical example is a monolayer‐stabilised nanoparticle (NP), in which an inorganic core is stabilised by a surface‐bound layer of molecular ligands. The properties of these multicomponent systems are defined both by the material, size and shape of the inorganic core and by the characteristics of the surface‐bound species Figure  a.3 FurtThe current strategies for in situ modification of NP‐bound molecules Figure  b, II–IVCovalent modification of NP surface functionality is an obvious attractive alternative and has indeed been intensively explored. However, typical examples rely on kinetically controlled reactions, often inspired by mild, robust and high‐yielding protocols developed for bioconjugation applications.Processes in which covalent bonds are formed reversibly under conditions of thermodynamic control have for decades been recognised as important in certain branches of chemistry, including carbohydrate stereochemistryCharacterisation of inherently dynamic and multicomponent chemical systems is a formidable challenge in any setting,Enabled by developments in scanning probe microscopy, potentially reversible covalent reactions have also been investigated for the construction of extended noncovalent and covalent atomically thin 2D networks at solid surfaces under vacuum,The emergence of surface‐confined dynamic covalent systems now suggests several compelling possibilities, including spatiotemporal control over the exchange process, leading to the prospect of creating surface patterns or achieving compartmentalised chemical behaviour spanning several length scales. These studies have helped to reveal the often considerable influence of the unique surface microenvironment when chemical processes are confined to interfaces.These contemporaneous developments for both nanomaterials and dynamic molecular systems now set the stage for extending dynamic covalent reactions onto nanosurfaces. Intermediate in size between macromolecules and extended surfaces, applying reversible covalent reactions to nanosurfaces allows the principles of equilibrium control to be applied to achieve responsive and adaptive behaviour on the nanoscale, while exploiting the precision and diversity of synthetic molecular structures. Working with nanosized chemical entities introduces several new challenges, but also opens up exciting possibilities for elucidating fundamental features of chemical processes taking place at interfaces that are just not available for extended substrates.In a handful of cases, cargoes have been attached to and then released from nanomaterials through formation and cleavage of the same covalent bond. For example, inspired by stationary phases developed for affinity chromatography, boronic acids have been associated with magnetic NPs and used to isolate and enrich polyhydroxylated biomolecules from biological samples followed by release for analysis by mass spectrometry.Taking place on sparsely‐functionalised irregular surfaces, direct characterisation of the molecular processes in such systems is a formidable challenge. These examples all exploit efficient interconversion of functional groups through high‐yielding condensation and hydrolysis reactions. Under each set of conditions, formation or cleavage of a covalent bond is essentially irreversible and is therefore conceptually similar to other covalent surface modifications.d≈200 nm) unilamellar vesicles assembled from egg phosphatidyl choline and 10 mol % of a membrane‐anchored thioester derivative 1  was observed, in contrast to the small monomeric and dimeric products formed in solution  bearing a hydrazone‐terminated surface monolayer  occurs rapidly and reversibly, achieving high surface‐saturation concentrations. Mixed boronate ester monolayers display pathway‐independent compositions that adapt to changes in the mixture of exogenously introduced diols, confirming that dynamic covalent exchange on the NP surface remains an equilibrium process under thermodynamic control , which is stabilised by a mixed monolayer incorporating approximately 10 mol % aldehyde ligand 12, with mixtures of simple primary amines, negligible NP‐bound imine formation was observed. However, on introducing short (16‐mer) oligonucleotides, selective uptake of amines from solution was observed. This could be explained by thermodynamic stabilisation of NP‐bound imines as a result of multivalent interactions between the imine‐functionalised NP surface and the oligonucleotide template. Significantly, the degree of functionalisation, and the composition of NP‐bound imine libraries, depended on the oligonucleotide sequence employed  functionalised with polymer brushes bearing approximately 10 mol % alkoxyamine side chains , subsequent quaternisation of the pyridine units rendered silica NP‐18−+P19Me dispersible in water. Reaction with the small‐molecule alkoxyamine 20 recovered both the XPS signature and solvent compatibility properties of the initial sample, consistent with a fully reversible dynamic covalent exchange process.In a recent study, Takahara, Otsuka and co‐workers prepared large silica NPs  constructed from soy bean lecithin and 25 mol % thioester 21 were treated with dithiol 22, resulting in covalent cross‐links between the vesicles , this offers a straightforward and rational approach to varying morphological parameters over a wider range than is possible using biomolecular systems. This modular strategy might also be extended to incorporate additional chemical, physical or structural features within the molecular linker design. The diversity of complementary and orthogonal dynamic covalent exchange processes points the way to yet‐more sophisticated assemblies and devices that incorporate several different DCNP building blocks, designed to be selectively reactive with each other and/or with specific species. The DCNP building block concept thus suggests several intriguing avenues towards responsive, reconfigurable and truly multifunctional hybrid nanomaterials in which both molecular and nanoscale components are precisely arranged, and combine to define emergent system properties.DCNP building block.One only has to look to biology to understand the potential for producing remarkable functional materials or complex chemical networks by nanoscale confinement of dynamic molecular systems on interfaces or within compartments. Advances in both synthetic and analytical technology are now allowing chemists to consider emulating some of these extraordinary systems, even if only at a comparatively rudimentary level. Dynamic covalent reactions offer unique advantages in their combination of specific and selective thermodynamically controlled reactivity, within structurally robust and unambiguous small‐molecule covalent structures that are synthetically and analytically tractable. Conferring dynamic covalent reactivity on the vast array of nanomaterials that can now be generated with increasingly precise control over chemical composition, size, shape and dispersity, will therefore establish a versatile new category of nanochemical synthon: the Whether on soft or hard nanosurfaces, combining the error‐correcting and environment‐responsive features of equilibrium processes with the stability and structural diversity of covalent chemistry amounts to a ‘best of both worlds’ solution to the problem of engineering surface functionality. The basic concept is largely independent of the nanomaterial itself, and so can ultimately be generalised across a range of materials, shapes and sizes. This is in stark contrast to ligand‐exchange strategies, which by definition must be specific for each different nanomaterial–molecule interaction; dynamic covalent exchange of simple units on the periphery of a stabilising monolayer can occur significantly faster under milder conditions and avoids the necessity for multistep synthesis of each ligand from scratch. The advantages of a nonbiomolecular approach include the ability to readily vary structural and chemical parameters, and operate under a wide range of conditions. The ever‐increasing number of well‐characterised dynamic covalent reactionsThe pseudomolecular nature of colloidally stable NPs—at least at the lower end of the size‐scale—means that although the analytical challenges are significant, they are not insurmountable. Several studies discussed herein have demonstrated that these systems can reveal direct insight into chemical processes occurring at interfaces, something that has proven extremely challenging in other settings. Consequently, DCNPs constitute a unique platform on which to study surface‐bound molecular behaviour. Understanding the subtle and complex network of interactions governing reactivity within NP‐bound monolayers will be crucial to arriving at rational and predictable synthetic methodologies for working with DCNPs, for example by quantifying and understanding the influence of surface confinement on reaction kinetics.The essential role that surface‐bound species play in defining a range of nanomaterial properties means that dynamic covalent modification heralds a powerful, environment‐responsive route to controlling system‐level behaviours, including physicochemical properties or microscopic and mesoscopic assembly structures. We have shown that this approach can be used to reversibly switch and tune NP solvent compatibility using very simple molecular exchange units,Mastering complex systems that arise through the interaction of dynamic chemical processes, reaction networks and structures is one of the next grand challenges facing synthetic, analytical and theoretical chemists in the coming decades;


Early Pleistocene examples as a point of departure, we emphasize the prominent role of viral and bacterial pathogens in oncogenesis and evaluate the impact of pathogens on human evolutionary processes in Africa. In the Shakespearean vernacular “what's past is prologue,” we highlight the significance of novel information derived from ancient pathogenic DNA. In particular, and given the temporal depth of human occupation in sub‐Saharan Africa, it is emphasized that the region is ideally positioned to play a strategic role in the discovery of ancient pathogenic drivers of not only human mortality, but also human evolution. Ancient African pathogen genome data can provide novel revelations concerning human‐pathogen coevolutionary processes, and such knowledge is essential for forecasting the ways in which emerging zoonotic and increasingly transmissible diseases might influence human demography and longevity in the future.The recent discovery that malignant neoplastic lesions date back nearly 2 million years ago not only highlights the antiquity of cancer in the human lineage, but also provides remarkable insight into ancestral hominin disease pathology. Using these  Most of these specimens comprise fragmented examples representative of incomplete skeletons, and only two display skeletal morphologies that relate to malignant tumorous development , while those that did survive long after the formation of tumours might, in some instances, have developed skeletal lesions  and extrinsic  risk factors in cancer development has been the subject of extensive scientific discussion  cells  comprise four well‐documented examples of associations between an aetiologic agent and the TP53 tumour mutation.Rudolf Virchow (1821–1902) first proposed the irritation hypothesis of carcinogenesis, positing that cancer development entailed the alteration of normal human cells. Following his observation of the inflammatory reaction in The fact that cancer incidence varies significantly amongst populations, organs and tissue types renders the prognostic accuracy of most  risk prediction models inadequate  to the lifetime risk of cancer development. On the contrary, the majority of cancers (70%–90%) can be ascribed to extrinsic environmental factors. Examples of environmentally induced cancers comprise colorectal cancer, with an estimated 75% of risk attributable to diet, malignant melanoma with 65%–86% of risk ascribed to excessive exposure to the sun and oesophageal cancers, in which case 75% are initiated by tobacco and alcohol abuse  classifies ~370 chemical compounds and microorganisms as “probably carcinogenic” to humans. Although the influence of infectious organisms on carcinogenesis requires considerable exploration , viruses  and parasitic microorganisms  are currently listed as recognized causal factors in oncogenesis. Several oncogenic viruses  are responsible for pathogen‐specific cancers , gonorrhoea (Neisseria gonorrhoeae), syphilis , HIV and trichomoniasis . The human sexual transmission pathway have also been implicated in the dissemination of re‐emerging pathogens such as Ebola virus disease  and bonobos (Pan paniscus) strengthens the idea that cross‐species mating also played an important role in the evolution of the great apes  that introgressed from an archaic population c. 35 ka , Denisovans (H. sapiens ssp. “Denisova”) and an as yet unidentified archaic group  is not uncommon  BMHs  and rinderpest (Morbillivirus), have been eradicated by vaccines. Many others, including polio (Picornaviridae) and influenza (Orthomyxoviridae) have not yet been eliminated  including two novel (Sudanese haemorrhagic fever and American and Polynesian Guillain–Barré syndrome) epidemic pathogens  analysis of archaeological human remains has gained increasing recognition  of aDNA from equid remains dated to ~700 ka  and Holocene (<12 ka) archaeological assemblages  are often misdiagnosed. Taphonomic alterations also mimic disease conditions which can induce interpretation errors (pseudopathologies), even for experienced palaeopathologists  and bioinformatic analyses of apDNA can possibly be used to anchor pathogen mutation rates and reconstruct viral and bacterial evolutionary processes. Second, and more controversially, it might also prove highly valuable in the development of new vaccines and, possibly, play a role in the discovery of novel pathogens that might pose significant future disease threats to humanity.H. pylori against ancient human migrations, the MRCA of H. pylori approximates that of anatomically modern humans. The genetic diversity of H. pylori also reflects other human demographic events, including the peopling of the Americas and Asia  affecting pathogen virulence and the ability of the bacterium to colonize and survive in flea intestines. While plague might have been less transmissible without arthropod vectors (fleas), it would still have been lethal as >90% of untreated cases of pneumonic plague is fatal. Prehistoric pathogen research can therefore contribute to our understanding of disease evolution by providing time‐stamped sequence data and by allowing the re‐evaluation of hypotheses regarding the extent of our coevolutionary history with pathogenic and commensal organisms  by Edward Jenner in 1796 , Neisseria meningitidis  and T. pallidum . The vaccine ST‐246 was developed using pathogenic Orthopoxvirus  genomic data and used to treat a child who developed life‐threatening eczema vaccinatum  from Siberian permafrost (Legendre et al., Vibrio cholerae (Baker‐Austin, Trinanes, Gonzalez‐Escalona, & Martinez‐Urtaza, The past provides a prologue for discussions regarding emerging diseases, whether it concerns the biological origins of a potential pandemic or its social repercussions Heymann, . DiseaseBecause of the paucity of aDNA sequences from Africa, these novel pathogen genomes will be highly valuable and decidedly revealing, providing novel revelations concerning human‐pathogen coevolutionary processes (Slatkin & Racimo,