Large-scale truck-involved crashes attract great attention due to their increasingly severe injuries. The majority of those crashes are passenger vehicle–truck collisions. This study intends to investigate the critical relationship between truck/passenger vehicle driver’s intentional or unintentional actions and the associated injury severity in passenger vehicle–truck crashes. A random-parameter model was developed to estimate the complicated associations between the risk factors and injury severity by using a comprehensive Virginia crash dataset. The model explored the unobserved heterogeneity while controlling for the driver, vehicle, and roadway factors. Compared with truck passengers, occupants in passenger vehicles are six times and ten times more likely to suffer minor injuries and serious/fatal injuries, respectively. Importantly, regardless of whether passenger vehicle drivers undertook intentional or unintentional actions, the crashes are more likely to associate with more severe injury outcomes. In addition, crashes occurring late at night and in early mornings are often correlated with more severe injuries. Such associations between explanatory factors and injury severity are found to vary across the passenger vehicle–truck crashes, and such significant variations of estimated parameters further confirmed the validity of applying the random-parameter model. More implications based on the results and suggestions in terms of safe driving are discussed. The economic impacts and safety hazards resulting from truck-involved crashes highlight freight transportation safety as a contemplative societal concern ,2. From Although most of the truck-involved crashes involve multiple vehicles, two-vehicle passenger vehicle–truck collisions often contribute to the majority (about 48%) of total fatal crashes according to the 2017 Fatality Analysis Reporting System (FARS) on fatal truck-involved crashes. For instance, in 2014, two-thirds of all police-reported crashes involved a truck and another vehicle , and 63%Some studies have successfully disentangled the complex associations between key factors  and the most severe injury outcomes in truck-involved collisions ,8,9,10. Quantify the associations between unsafe driving behaviors and injury severity outcomes in passenger vehicle–truck collisions. Specifically, the intentional and unintentional driving actions of truck and passenger vehicle drivers.Explore unobserved heterogeneity in the associations of injury severity with unsafe pre-crash behaviors, while controlling for the driver, vehicle, and roadway factors.The present study contributes by focusing on two objectives:An explicit investigation of drivers’ unsafe pre-crash actions is likely to allow us to develop actionable safety improvement strategies. In order to achieve the objectives, sophisticated fixed- and random-parameter ordered probit models are estimated by using real-world police-reported crashes, which allows for the unearthing of important and embedded relationships in crash data. The content of the paper is original and timely given the enormous costs sustained by society in consequence to passenger vehicle–truck collisions, and the implications of safety concerns for occupants of passenger vehicles. Given the considerable costs imposed by truck-involved collisions, several researchers have investigated such collisions and the key factors that may be associated with injury outcomes. In this section, previous studies are synthesized with the specific focus on methodological approaches that are used for establishing associations between different crash characteristics, unsafe pre-crash behaviors, and injury outcomes in passenger vehicle–truck collisions. A broad spectrum of studies investigated the associations between several factors such as collision types, roadway types, vehicle types, and injury outcomes in passenger vehicle–truck collisions ,17,18,19The role of driver actions in truck-involved crashes has also received considerable attention ,16,23,27The aforementioned studies did not focus primarily on investigating a broad range of unsafe pre-crash driving behaviors, intentional and unintentional improper actions of either truck driver or passenger vehicle driver, and the implications on most severe injury outcomes of passenger vehicle–truck collisions. An explicit investigation of drivers’ unsafe pre-crash actions can help in developing actionable safety improvement strategies for passenger vehicle–truck collisions. Furthermore, due to the complex crash data structure and different unobserved crash-, vehicle-, and driver-related factors, the associations between key driving behaviors and injury outcomes may vary significantly across different crashes. Ignoring the possibility of varying relations between key explanatory factors and injury outcomes can neglect important information embedded in passenger vehicle–truck crash data . The stuThis paper used the 2013 Virginia Police Crash Reports obtained from the Virginia Department of Transportation. The database is comprehensive and well-organized containing records of crashes occurring across Virginia. For this study, three files are extracted from the database and linked together in order to obtain the crash, vehicle, and person-level information involved in passenger vehicle–truck collisions. Specifically, the crash file contains information on variables describing the crash, crash time, roadway characteristics, and collision type—the vehicle file contains information on vehicles such as vehicle body type, and the person file contains information on occupants (including driver) such as age and gender, level of injury sustained, and other driver-related factors. All three files are linked together through a unique crash identification number. Among 121,601 crashes documented in the database, 7501 are truck-involved crashes, and 4926 crashes are two-vehicle passenger vehicle–truck crashes. Given the focus of this study (as explained earlier), two-vehicle passenger vehicle–truck collisions are extracted , which accounts for a significant 66% of the total truck-involved collisions. Given the national average of 63%, the sample size at hand is reasonably representative. There are nine combinations of different (truck and passenger vehicle) driver actions that are shown in In terms of injury severity, this study utilized the most severe injury among all the occupants in both vehicles as the injury severity level of the crash. As reported in the police crash report forms, five levels of injury severity are observed: killed, serious injury, minor injury, no apparent injury, and non-injury (property damage only). However, due to the limited number of crashes with fatalities, and in order to facilitate the analysis, the injury severity scale is categorized into fatal/serious injury, minor injury, and no injury. Several studies in the past have re-categorized injury severity scales due to limited cases with fatalities . Finally, to analyze the associations between unintentional and intentional driver actions (either the truck driver or passenger vehicle driver), the driver actions reported in VDOT police crash report forms are classified into four categories:Action 1: Passenger vehicle driver undertook no improper action while truck driver undertook an intentional improper action.Action 2: Passenger vehicle driver undertook no improper action while truck driver undertook unintentional improper action.Action 3: Passenger vehicle driver undertook intentional improper action while truck driver undertook no improper action.Action 4: Passenger vehicle driver undertook unintentional improper action while truck driver undertook no improper action.To have a general map of those actions, the intentional actions include behaviors such as speeding, following too close, wrong place or no right-of-way, disregard of officers, signals or signs, and so forth; the unintentional actions include driver behavior that related to driving technique inadequate, failing to maintain proper control, avoiding animals or object and other improper actions, and so forth. Note that the classification scheme adopted in this study is consistent with Liu et al. , who invAn ordered probit modeling framework is used in this study due to the ordinal nature of the response outcome . The modNote that the above framework implies an unduly restrictive assumption of constant parameter effects across sampled observations. For instance, one coefficient is estimated for each explanatory factor at times when the associations between explanatory factors and injury severity may vary across sampled observations due to the presence of several observed and unobserved factors . In the In this study, the random-parameter ordered probit model is estimated by simulated maximum likelihood estimation, and by using 200 Halton draws as recommended by other studies . For ranFinally, after model estimation, the signs of parameter estimates are of great importance. A positive sign shows an increase in the probability of the most severe injury severity and a decrease in the probability of the least severe outcome and vice versa for negative parameter estimates . The coeribution ,37. The present study analyzes 3774 passenger vehicle–truck crashes, which involve 7548 vehicles and 7871 individuals. Regarding the key explanatory variables, Based on the descriptive statistics, the data seem to be of reasonable quality. In 37.5% of the collisions, passenger vehicle driver undertook no improper actions while truck driver undertook an intentional improper action (action 1), as opposed to 41.3% of collisions in which truck driver undertook no improper actions and passenger vehicle driver undertook intentional improper action (action 3). Almost 30% of passenger vehicle–truck collisions were angle collisions, and approximately 21% of them occurred between 01:00 and 08:00. The average total number of injured is 0.388 in all collisions . Explanatory variables are identified by developing the simple correlation matrices of key factors with injury severity of the most severely injured person in passenger vehicle–truck collisions. This helped in identifying potential factors related to driver actions, collision types, roadway types, driver conditions, demographics, and temporal characteristics. Next, a series of fixed-parameter ordered probit models are estimated for injury severity in the collision. Most variables were either statistically significant at a 95% confidence level or theoretically important and retained for subsequent analyses. The results of the final fixed–parameter ordered probit model are presented in The incorporation of random parameters resulted in the overall improvement of fitness compared with the fixed-parameter model . MoreoveTo facilitate discussion of estimated models, the explanatory factors  are cateRegarding driver actions , action Interestingly, if the passenger vehicle driver is fatigued or asleep in a collision, the injury severity is higher. Results show that fatigued driving is associated with injury severity at a 90% confidence level. While the parameter estimates for this variable are found to be fixed, the marginal effects obtained from fixed- and random-parameter models reveal differences. The average marginal effects for fatigue  show thaIn terms of driver demographics, the age of the passenger vehicle driver is found to be a normally distributed random parameter with a mean of −0.187 and a standard deviation of 0.232. Passenger vehicle drivers aged 20–29 years are negatively associated with injury outcomes. This finding is in agreement with other studies . In thisCompared with interstates, principal arterials, minor arterials, collector and local are all expected to be negatively associated with severe injury outcomes. Local roadway systems are more likely to be associated with less severe crashes, compared with other roadway types. Notably, principal arterials and minor arterials both resulted in parameter randomness. The significance of principal arterial and collector are improved from 95% in the fixed model to 99% in the random model. Principal arterials are normally distributed with a mean of −0.433 and a standard deviation of 0.849 , suggestOut of all collision-type factors, head-on and angle collisions are found to be statistically positively associated with the most severe injury outcomes in passenger vehicle–truck collisions, while sideswipe-same direction collisions are negatively associated with the injury severity. Note that both head-on and sideswipe-same direction collisions are significantly associated with injury severity at a 99% level of confidence in both models. The significance of angle collisions is found to be enhanced in the random-parameter model . Numerous studies have found positive associations between collision types (head-on and angle) and injury outcomes. However, from a passenger vehicle–truck collision perspective, the injury outcomes may be more severe, which is potentially due to the enormous physical momentum of colliding trucks . As suchThe study investigates the associations between unsafe driver actions and injury outcomes in passenger vehicle–truck collisions by using the real-world police-reported crashes, which has many intrinsic limitations ,39. For This study contributes by quantifying associations of intentional and unintentional improper actions of truck and passenger vehicle drivers and driver condition with injury severity outcomes in passenger vehicle–truck collisions. The study explicitly accounts for unobserved heterogeneity and finds that some of the correlates have both positive and negative associations with injury severity. Rigorous fixed- and random-parameter ordered probit models are estimated using 2013 state-wide passenger vehicle–truck collision data from the Commonwealth of Virginia. Significant efforts went into processing the raw data and linking different databases for collecting important information on the crash, vehicle, and driver-related factors. The model results showed that compared with fixed-parameter and random-effects ordered probit models, the random-parameter model provides a superior fit to data at hand and provides fuller information regarding the relationships between key factors and the most severe injury outcomes. Compared with truck occupants, passenger vehicle occupants are six times more likely to sustain minor injuries and ten times more likely to suffer serious/fatal injuries. All else being equal, intentional improper actions and unintentional improper actions of the passenger vehicle drivers are both positively associated with higher injury severity, while the unintentional actions of the truck drivers are negatively associated with injury severity in passenger vehicle–truck crashes. Passenger vehicle driver-related factors such as driving under the influence of alcohol and fatigue are associated with higher injury outcomes. Importantly, compared with other times of day, passenger vehicle–truck collisions occurring late at night and in early mornings (01:00 and 08:00) are associated with higher injury severity in such collisions. From a behavioral perspective, a specific taxonomy of driver errors, whether intentional or unintentional, should be targeted given that passenger vehicle driver errors are significantly associated with higher injury outcomes. The safety literature shows numerous examples where intentional errors are associated with higher injury outcomes. However, in the case of passenger vehicle–truck collisions, unintentional errors are also of great concern. Driver awareness and training programs such as educating passenger vehicle drivers about driving carefully in the vicinity of trucks may also target driver errors that increase injury severity. Regarding driver behaviors and conditions, driving while fatigued or falling asleep and collisions occurring late at night and in early mornings are high-risk factors associated with injury severity. Countermeasures  should be considered to minimize such risk factors. Finally, from a methodological standpoint, the study results imply that addressing unobserved heterogeneity is important in injury analysis of such collisions. Ignoring unobserved heterogeneity can mask important information embedded in data, which may affect the quantification of effects of risk factors and hence the development of appropriate strategies. Due to the limitations of this study, further work will be performed to examine more comprehensive crash databases in order to validate the findings by exploring the effects of underreporting issues of data quality.


Adult stem cells are excellent cell resource for cell therapy and regenerative medicine. Dental pulp stem cells (DPSCs) have been discovered and well known in various application. Here, we reviewed the history of dental pulp stem cell study and the detail experimental method including isolation, culture, cryopreservation, and the differentiation strategy to different cell lineage. Moreover, we discussed the future potential application of the combination of tissue engineering and of DPSC differentiation. This review will help the new learner to quickly get into the DPSC filed. Stem cell (SC) was firstly termed and reported in the literature in the nineteenth century by Alexander Maksimov . During The two shining points of SC are its pluripotency and differentiation ability, so as DPSCs. The DPSCs have high proliferative potential which shows a strong cloning ability, originating from dental pulp genesis Fig.  83, 89], 8983, 8The most obvious advantages of DPSCs are its highly proliferative capacity and differentiation possibility into various cells including epithelial cells, vascular cells, adipocytes, odontoblasts, osteoblasts, neuronal cells, and muscular cells. As the dental pulp is one of the major sites when considering bone marrow as the choice for hematopoietic stem cell collection, DPSCs are considered to be the ideal source for clinical application and tissue engineering . DPSCs’ In order to better understand DPSCs’ character for further applications, many strategies have been developed to isolate DPSCs from adult dental pulp , 80. OneIn adult stem cell isolation, the key points are dissociation methods and selection markers. The dissociation method led the vitality of the stem cells, while the selection markers led the purity. Two main approaches have been applied for DPSCs dissociation, one is directly from pulp tissue (DPSC-ED) and the other one is outgrowth from tissue explants (DPSC-OG). Over 20 different enzymes and their combinations were considered during the DPSC dissociation methods. Collagenase I associated with dispase and collagenase alone were the most frequently used methods . A recenUnlike the dissociation method, the DPSC culture method still needs further development. The most common basal medium is α-MEM followed by DMEM. For the further application purpose, Serum-free media have been continuously tested for stem cell culture. Guo and his group established a chemically defined serum-free culture system for human dental pulp stem cells and found the serum-free Essential 8 medium might be applicable for hDPSCs culture in the future . HoweverAs mentioned before, DPSCs’ cryopreserved character is the necessary advantage for cell-based therapy. Gianpaolo Papaccio and his group demonstrated DPSCs and their osteoblast-derived cells can be long-term cryopreserved . LindemaDifferentiation ability is another key character of stem cell, so does DPSCs. As one kind of adult stem cells, the most key differentiation potential is the purpose tissue or cells itself. To DPSCs, it means odontoblasts. After transplanting into immune-compromised mice, human DPSCs will automatically differentiate into the dentin-like structure with odontoblast-like cells . By seedDPSCs have also been proved to differentiate into other cell types including all three germ layers. The detailed information of differentiation methods and checking notes were shown in Table The ectoderm or endoderm lineage cells differentiated from DPSCs have also been reported. DPSCs express high expression of various neural crest-related and neural crest developmental genes since they are derived from neural crest origin , 52. DPSA hepatic lineage of cells has been differentiated from human dental pulp cells although the purity was not very high . More anDPSCs can be used in various aspects such as tissue engineering and regenerative medicine owing to their high proliferative and multilineage differentiated ability , 58. It The first successful example is treating spinal cord injury (SCI) which leads to sensation loss and extremities paralysis. Comparing with BMSC and AMSC, DPSCs could enhance the survival rate of endogenous glia and neurons  owing toMoreover, DPSCs may involve in deciduous teeth root reabsorption process . PierdomExcitingly, DPSCs have been used in vivo and reported in many articles. Two systematic reviews , 53 wereWhile the shortage of traditional cell culture limits the application of stem cell-based cell therapy, tissue engineering strategies were used to enhance DPSCs in vitro function. By mimicking the physicochemical and biological signal niche, combining the engineering and material methods , 54, andUsually, the basic tissue engineering method includes scaffold reconstruction, hydrogel matrix support, and artificial patterning design. These three methods always support each other to develop enhanced complex both in tissue structure and function. Limited by the weak mechanical properties of the hydrogel, scaffolds have usually been fabricated as the framework for cellular behavior and function. For regeneration medicine use, the first consideration of the scaffold material should be the character of non-toxic, less side effects, high biocompatibility, and low immunogenicity. For the engineering tissue preparation, the scaffold material needs to be extensive and the preparation process should be simple and repeatable, as well as environmentally friendly. Undoubtedly, all the above pose-specific problems and challenges for the development of tissue engineering scaffold materials indicate the direction.Choosing biocompatible materials and designing mimic device will help to enhance cell interaction and stimulating spontaneously regeneration to format self-regulating tissue function, instead of artificially constructing complexity of living tissues ex vivo. The 3D geometrical features, mechanical property, surface feature, and other biocompatibility of materials influence cell adhesion, proliferation, differentiation, and pluripotency maintenance. The different cells in different surrounding materials are able to regulate self-gene expressions and secrete new ECM which will lead the other cells fate determination . A well-Combined tissue engineering with DPSC, some approaches towards the regeneration medicine application have already been reported. Base on the serious of the disease and DPSCs’ characters, most of the applications focus on neurology, tooth or bone reconstruction, and angiogenesis, liver, esophageal, or bladder regeneration. Here, we list the examples for hDPSCs combined with tissue engineering in neuro-, bone, and dentin-pulp regeneration.A study of 3D Floating Sphere Culture System provides a respectable microenvironment for hDPSCs to preserve their neuronal properties significantly compared to osteogenic and myogenic commitments . If the Previous studies have been well demonstrated the osteogenic differentiation capacity of DPSCs, including high expression of bone-specific markers . ScaffolRoot canal therapy is a common treatment for trauma and infection of adult pulp. After root canal therapy, the entire pulp was removed and often followed by tooth fracture and finally lead to loss of teeth. It is noteworthy that DPSCs have the capacity to generate dentin that rendered dentin-pulp regeneration possibility . The proMoreover, special growth factors and scaffold materials are important factors for improving functional effectiveness in dentin-pulp TE. According to the report, DPSCs that are transplanted with stromal cell-derived factor 1 (SDF1) in a collagen scaffold could complete pulp regeneration in a canine pulpitis model . In addiAs a promising cell source for numerous and varied regenerative medicine applications, DPSCs become one of the target adult stem cells which need to further understand and develop. Although some achievements have been made in the study of DPSC, several challenges remain to be solved. For example, as the main problem in all kinds of stem cells transplantation, low survival rates, especially low long-time survival rates, were still a key concern for whether the recover function really comes from DPSCs’ tissue function rebuild or secretion effect , 55. Mul


Food purchasing and consumption behaviors have implications for nutrition and obesity. Food retail environments, in particular, shape customer food choices and energy intake. The marketing literature offers insights about how public health practitioners can work within food retail environments to encourage healthy food choices. We reviewed experimental studies in the marketing literature to examine factors influencing customer purchase intentions and choice for food products in retail stores. Database searches were conducted in February 2016 for original, empirical articles published in English from 2000–2015 in marketing journals. Each research article included at least one experimental design study conducted in a real or simulated retail environment with purchase intentions or choice of food products as an outcome variable. Backward and forward reference searches were conducted for articles meeting inclusion criteria. Narrative synthesis methods were used to thematically group and summarize the findings of forty-one articles that met inclusion criteria into three categories: shelf display and product factors, pricing and price promotion factors, and in-store and customer decision-making factors. This research contributes to the literature by providing specific and actionable approaches that can increase/decrease customer purchase intentions and choice for food products in retail environments. Translating marketing strategies into public health applications can provide recommendations for future intervention research and policy related to customer food purchasing behavior. In the United States, an estimated 35% of adults and 17% of children are obese ,3,4. ConFood shopping behavior continues to evolve, and food retail environments are increasingly more complex, requiring a more in-depth understanding of customer behavior . Food shExisting evidence on healthy food retail strategies indicates an important gap in knowledge on how best to promote healthy food choices and purchases in food retail environments . ProgresThe primary references in this scoping review were peer-reviewed articles published in marketing journals studying purchase intentions and/or choice in food retail environments. The American Marketing Association defines marketing as “the activity, set of institutions, and processes for creating, communicating, delivering and exchanging offerings that have value for customers, clients, partners and society at large” . UnderstThe authors employed a three-step search strategy from February to July 2016, utilizing methods developed by Cooper (1998) . For theFor the database searches, an enhanced concept-to-operation correspondence was captured via multiple operationalism recommended for literature reviews . We usedSearch results were limited to articles (1) written in English, (2) published between 2000–2015, (3) original research conducted in any part of the world, and (4) published in peer-reviewed journals in marketing ,14. ResuArticles published in marketing journals frequently contain multiple studies in one article. Data extracted from relevant studies that met inclusion criteria included manipulated variables and conditions, type of experiment, outcome variables, and key findings. In order to summarize and disseminate the research findings, narrative synthesis was used to thematically group and report findings in the manuscript and a table was created that captured detailed information from each article and serves as a reference for future research .This scoping review focused on studies that employed experimental research designs, in which individual factors are manipulated in order to study and measure the effect of each manipulation on the outcome of interest . Twenty-Each article included an average of three studies (range: 1–5); the majority of articles had one or two studies that met inclusion criteria. Please refer to Customer choice and purchase intentions can be influenced by product location on the shelf, the appearance of the products on the shelf, the brands available, and product attributes. Shelf display and product factors can capture customers’ attention and serve as sources of information for customers when making purchase decisions ,17,18,19Customer choices are also associated with the inferences customers draw when looking at shelf displays. Product display factors, such as shelf-based scarcity  can create positive inferences regarding the popularity and quality of the products on display, leading to higher purchase intentions for those products ,21,22. IBrand familiarity and availability can also impact purchase intentions and choice. Brand familiarity is important as customers are more likely to choose familiar over unfamiliar brands . CustomeProviding health information on product packaging can positively or negatively influence purchase intentions and choice based on customer knowledge ,30. HighFront-of-package labeling systems can also provide customers with information that can influence decision-making. Customers use front-of-package labeling systems, including reductive labels  and evaluative labels  differently. Evaluative labels are more beneficial when customers are comparing products, while reductive labels are more effective when evaluating a single product . FinallyFood samples can impact the healthfulness of products purchased ,35. OffePrice and promotion information, and how it is presented, can influence purchase intentions and choice. Unit price information can lead customers to choose products with lower unit prices , since iPrice promotions can negatively influence sales of products after the sale period has ended. Promotions that offer large percentage off (versus cents off) discounts negatively influence customers’ purchase intentions for the brand after the sale . AdditioCustomers’ responses to price promotions are based on individual differences, including their decision-making styles  and regulatory focus . Analytical decision-makers focus on both utilitarian and hedonic benefits, and intuitive decision-makers focus on hedonic benefits, influencing likelihood to buy . AdditioBoth intrapersonal factors and in-store environment factors can impact the customer’s experience while in the store and shape customer purchase intentions and choice ,53,54,55The processes that customers engage in while shopping may also increase unhealthy choices. When customers make multiple choices in a row when grocery shopping , their self-control weakens, leading to unhealthy subsequent choices . For cusStore environment factors also steer behaviors. When customers feel confined  they make more varied choices , and a sThe findings of this review build on existing intervention research in the field of public health ,59,60,61Some intervention strategies can begin before the customer enters the store and others can be implemented to facilitate healthier choices while shopping. Intervention strategies can focus on educating customers on how to prepare for the shopping experience. The United States Department of Agriculture (USDA) Supplemental Nutrition Assistance Program Education (SNAP-Ed) teaches program participants about nutrition and how to stretch their food dollars. Existing recommendations include planning ahead to know what is needed, making a shopping list, and knowing how much is available to spend . Our finStore-based interventions have focused on point of purchase information, price promotion strategies, and healthy food accessibility and availability ,59,60,61Past intervention research has implemented pricing strategies  ,60,61; hResearch has addressed ways of nudging consumers toward healthier choices through product placement, increasing the salience of food accessibility and availability . The finInsights on the effectiveness of nutrition labeling on packaging also raise important policy implications. In May 2016, after 20 years of the same nutrition label, the Food and Drug Administration (FDA) released a new nutrition label design . The newA key strength of the current review is the inter-disciplinary approach to understanding food purchases and health. By reviewing articles in the marketing discipline, our goal is to motivate evidence-based marketing practices to be translated into public health applications. This project was also a collaboration between public health and marketing researchers. Public health researchers provided critical inputs during the research conceptualization and manuscript development process to contextualize marketing insights in a public health framework. Marketing researchers, in turn, lent their domain expertise during data extraction and synthesis and during manuscript preparation. The current review maximizes the opportunity for the inter-disciplinary exchange of ideas and knowledge to achieve better health outcomes for communities.The search for articles was limited to peer-reviewed journals in English, which may introduce a publication bias if published articles do not represent marketing scholarship investigating choice and purchase intentions. Additionally, the search for articles was conducted in 2016, so the review does not include articles that may meet inclusion criteria published after the search dates. In order to synthesize findings to understand consumer behavior, we limited the review to include laboratory and field experiments with purchase intentions or choice of food products as the outcome variables. In doing so, a number of articles with relevant outcome variables and settings were excluded because they were based on secondary panel data, sales/scanner data, survey/interview data, qualitative data, and/or did not include food products. While findings from these studies may further expand understanding of customer choice and purchase intentions, the emphasis on laboratory and field experiments as a first step allowed us to focus on understanding how controlled manipulations directly affect these outcome variables. Additionally, we are unable to relate the findings to socio-economic disparities. Future public health interventions can assess the implications of these findings given different socio-economic contexts. Despite these limitations, the strengths of this review include drawing a bridge between marketing insights and actionable strategies for healthy food-related interventions and identifying future avenues of research for obesity and nutrition interventions.While findings reveal how different factors affect customer buying behavior, future studies must explore how these translate into long-term and sustainable consumer behavior change, including changes to actual consumption and repeat purchases over time. Understanding how sampling, nutrition labeling, shelf and product displays, and pricing and promotions impact behavior in the food retail environment may help policy makers understand and prioritize strategies to promote customer choice of healthy items. A series of longitudinal, randomized controlled trials to examine the interaction of these variables in food retail environments over time would allow researchers to tease out the most important factors, and make evidence-based policy recommendations based on the most influential and cost-effective factors. Future studies must examine varied theoretical approaches as well as culture, geographic location, socioeconomic status, and other demographics and how they may influence customer choice in a variety of large and small food retail settings. Finally, the marketing literature provides theories that may be useful in further understanding customer behavior in food retail environments. Future research can review and incorporate consumer behavior theories into intervention development.


Technological advances are continually improving our ability to obtain more accurate views about the inner workings of biological systems. One such rapidly evolving area is single cell biology, and in particular gene expression and its regulation by transcription factors in response to intrinsic and extrinsic factors. Regarding the study of transcription factors, we discuss some of the promises and pitfalls associated with investigating how individual cells regulate gene expression through modulation of transcription factor activities. Specifically, we discuss four leading experimental approaches, the data that can be obtained from each, and important considerations that investigators should be aware of when drawing conclusions from such data. To perform essential biological functions and respond to changes in their microenvironments, cells systematically change the expression levels of process-relevant genes, while maintaining homeostatic expression of so-called housekeeping genes. For example, after detecting lipopolysaccharide (LPS), a bacteria-derived substance and molecular indicator of infection, macrophages alter the expression levels of several thousand genes, while maintaining baseline expression of other genes . We stilIn investigations of TF dynamics in single cells, TFs are often expressed as fluorescent fusion proteins and imaged via live-cell fluorescence microscopy. Imaging enables real-time recording of stimulus-induced changes in single cell TF abundance, nuclear localization, and residence time ,13,14. PChanges in the population-level expression of specific target genes have been correlated to single cell dynamics of TFs, including nuclear factor κB (NF-κB) ,17,18,19Single-molecule RNA fluorescence in situ hybridization (smRNA-FISH) is a molecular technique characterized by its ability to detect low levels (<5 copies per cell) of endogenous RNA transcripts of interest in individual cells ,33. It rsame single cells over time. Instead, only a single endpoint snapshot measurement of gene expression can be obtained per cell, after TF dynamics have been continuously imaged in each cell  offers a uniquely powerful advantage which is orthogonal to smRNA-FISH, as this approach enables an unbiased profiling of gene expression. It measures the levels of endogenous RNA transcripts expressed in individual cells in a genome-wide manner by next-generation sequencing technology ,41,42,43Unfortunately, scRNA-seq possesses the same pitfall as smRNA-FISH because it is also limited to providing only one single static snapshot of gene expression per cell B. scRNA-Measuring the expression of fluorescent reporter proteins in single cells is arguably the most information-rich approach for investigating the impact of TF dynamics on gene expression. Fluorescent reporter proteins, typically enhanced green fluorescent protein (EGFP) or alternatives, are expressed from customized gene expression cassettes introduced into cells by various means. The cassettes can be integrated into chromatin in cell lines and in vivo systems at random  or at spsame single cells by using fluorescent proteins with distinct excitation and emission wavelengths for the TF fusion protein and the reporter (There are several advantages of using fluorescent reporter proteins to investigate single cell gene expression, especially as it relates to TF dynamics. First, expression levels of fluorescent reporter proteins can be measured in real-time at high temporal resolution, over many hours or even days B, withoureporter A,B. CaptStudies utilizing fluorescent reporter proteins to quantify gene expression and TF dynamics in the same single cells ,29,30,31The use of fluorescent reporter proteins to measure single cell gene expression does have limitations. Unlike smRNA-FISH and scRNA-seq, the fluorescent proteins do not report expression of endogenous RNA transcripts. To date, we are unaware of studies that have investigated TF dynamics-mediated regulation of endogenous gene loci using this approach. In addition, due to constraints associated with the overlap of fluorescent protein excitation and emission spectra, only a few reporter proteins can be simultaneously imaged as readouts for expression of different genes. Detection of signals from fluorescent reporters is likely to be not as sensitive as smRNA-FISH for low-abundance transcripts. Imaging very low-abundance fluorescent reporters may require using an increased laser power, which can result in cellular toxicity if not addressed by adjusting other image acquisition parameters.A marked alternative to assessing gene expression through measurements of RNA transcripts or intracellular protein abundance is measuring the amount of induced proteins that are secreted from single cells. Different research groups have engineered microfluidic devices capable of culturing single cells in isolation, stimulating them individually, and then capturing and quantifying the levels of specific proteins that are secreted by each single cell via antibody-based immunoassays ,57,58,59The fact that separate biochemical processes underlie the regulation of gene expression and protein secretion is probably the most significant pitfall associated with utilizing secreted proteins as measures of gene expression. It is not difficult to envision a case where single cell gene expression correlates with the dynamics of a TF, but the kinetics and extent of single cell protein secretion do not. Another caveat of the approach, which is shared by scRNA-seq, is the requirement for strict isolation of single cells in individual culture wells or channels. When interpreting data or comparing results with those of other studies, these points should be taken into consideration. Advances in microscopy, high-throughput sequencing, and microfluidics are steadily making the study of single cell TF dynamics more tractable. However, many unresolved and technically challenging issues remain regarding the fundamental aspects of TF dynamics and their role in gene regulation. Each of the approaches to measuring single cell gene expression discussed here, when coupled with imaging TF dynamics, has its own benefits and pitfalls. Any of the given methods may be the most appropriate depending on specific research needs. Nonetheless, we argue that the use of fluorescent reporter proteins is the technique which may well be the best option currently, being the only approach that provides real-time data in the same single cells. Such information-rich data can be used to determine per-cell correlations between TF dynamics and gene expression, which provides critical information for mechanistic modeling of the relevant regulatory processes. We look forward to improvements in existing technologies and the development of new methods that will facilitate our abilities to simultaneously measure real-time TF dynamics as well as expression of many endogenous genes in the same cells, in vitro and in vivo.


Advances in nanoparticle (NP) production and demand for control over nanoscale systems have had significant impact on tissue engineering and regenerative medicine (TERM). NPs with low toxicity, contrasting agent properties, tailorable characteristics, targeted/stimuli-response delivery potential, and precise control over behavior  have made it possible their use for improving engineered tissues and overcoming obstacles in TERM. Functional tissue and organ replacements require a high degree of spatial and temporal control over the biological events and also their real-time monitoring. Presentation and local delivery of bioactive  and contrast agents in a controlled manner are important implements to exert control over and monitor the engineered tissues. This need resulted in utilization of NP based systems in tissue engineering scaffolds for delivery of multiple growth factors, for providing contrast for imaging and also for controlling properties of the scaffolds. Depending on the application, materials, as polymers, metals, ceramics and their different composites can be utilized for production of NPs. In this review, we will cover the use of NP systems in TERM and also provide an outlook for future potential use of such systems. Due to many drawbacks of tissue and organ transplantation such as limited donor availability, the need for immunosuppression and insufficient success rate (rejection of the transplant), there is an increasing demand in tissue engineering and regenerative medicine (TERM) solutions which is a rapidly growing multidisciplinary field. It has merged the biological, material and engineering sciences to develop and manufacture artificial structures that resemble the native tissue/organ not only as implantable systems but also as model, miniaturized organs  can be described as a colloid of nanometer sized particles of gold. Colloidal gold solutions present different properties compared to the bulk gold, for example, their optical property due to their unique interaction with light  absorbing plasmonic AuNPs took place at the nuclear region of the cells  coated AuNPs (30_nm) through binding it with nuclear localization signal (NLS) peptides together with arginine—glycine—aspartic acid (RGD)  through an antibody, for targeting tumor cells. The antibody fragment recognizes EGFR on cancer cells  conjugated AuNPs have been reported to be efficiently uptaken by human mesenchymal stem cells (MSCs) and reduce the overproduction of reactive oxygen species in them at low dosage of TEMPO  (50 ng/ml) enhanced cell migration on the scaffold of FN-AuNPs via signaling pathway of matrix metalloproteinase (MMP)/ endothelial nitric oxide synthase (eNOS). MSCs have shown higher antithrombotic activity, better endothelialization and higher expression of CD31 and alpha-smooth muscle actin (α-SMA) with FN-AuNPs coated catheters implanted compared to control groups.Chen et al. have reported the impact of the AuNPs on MSCs for vascular tissue engineering  into metallic silver (Ag0), NPs will be formed through nucleation followed by the growth. In the past few years, biological methods using microorganisms such as bacteria or, eukaryotic fungi or plants to reduce silver ions, have emerged to synthetize AgNPs (Silver nanoparticles (AgNPs) can also be described as a colloid of nanometer sized particles of silver and are one the most widely used metallic NPs in biomedical field mainly for their antimicrobial properties  (PVA), PCL, gelatin, chitosan-alginate and cellulose acetate scaffolds. All the resulting scaffolds exhibited strong antimicrobial activity.in situ in the structure using reducing agents . These different scaffolds can be produced in different formats including bulk materials, electrospun fibers, fibers mats, nanofibrous or porous scaffolds  are basically comprised of inorganic compounds, besides metals, metal oxides, and metal sulfides and they can be used in production of nanoscale materials of various shape, size, and porosity  in the sol–gel procedure through immersion in simulated body fluid  with SiO9 Jones, . Bioglas9 Jones, . Various9 Jones, . nBGC ca9 Jones, . Effect Antibacterial and angiogenic properties and excellent bioactivity of nBGs have made them as a suitable candidate for dentin regeneration applications. Incorporation of boron modified nBGs in the cellulose acetate/oxidized pullulan/gelatin-based constructs has shown promising results for dentin regeneration through increase in cellular viability, Intracellular calcium deposition (ICD) and ALP activity while keeping the boron ion released below toxic level  in the defect area after 8 weeks of implantation compared to control groups. In another study, multifunctional poly(citrate-siloxane) (PCS) elastomer based nBGs were developed for bone tissue engineering . Additionally, it enhanced osteoblastic differentiation of these cells and has also decreased the in vivo inflammatory response toward the biomaterial. Various other applications have been investigated using nBGs for bone tissue engineering including copper containing nBGs in gelatin coated scaffolds , calcium sulfate hemihydrate, octacalcium phosphate and biphasic calcium phosphate. HAp is major component (inorganic) of natural bone and under neutral or alkaline conditions it is the most stable form of phosphate salts. These materials have been applied in orthopedics such as bone substitutes  based composition which include variety of materials such as hydroxyapatite (HAp), calcium aluminate, tricalcium phosphate, calcium phosphate dicalcium phosphate dehydrate, calcium carbonate  have been doped into nHAp via microwave-assisted synthesis and under various operating voltages significant enhancement in the contrast efficiency was observed /selenite , zinc oxide (ZnO) are utilized for different medical applications as they show positive interactions with body tissues. TiO2 NPs can be synthesized with different manufacturing processes including hydrothermal, solvothermal, sol-gel process and emulsion precipitation methods   and a functionalized MTNs with a phosphate-containing fluorescent molecule  have been recently synthesized. FMN-MTNs were used as a satisfactory intracellular bioimaging agent , and they were further observed inside the MTNs without much leaching  which are widely studied in biomedical field because of their lower toxicity. Co-precipitation is used as most conventional method for synthesizing Fe3O4 or γ-Fe2O3  can be utilized for cell imaging. Mini emulsion/sol-gel and polyol techniques were used for synthesizing SMNCs. They were prepared by coating with polyetherimide, citric acid or silica. These SMNCs possessed very high sensitivity toward magnetic resonance, and had no adverse effect on cell viability  are iron oxide NPs,  to reduce anxiety-like behavior of rats through binding to corticotropin releasing factor type 2 receptors  with no impact on locomotion.One of the exciting applications of TERM is the neuroregeneration and nerve tissue engineering which could improve life quality of the patients with nerve injuries. Various studies have been conducted to deliver growth factor conjugated MNPs to cells, monitoring the fate of MNPs or stimulate the cells with MNPs , formulation, size, shape, surface chemistry and charge, porosity, mechanical strength, solubility, degradation rate and so on are among the features which can be adjusted for versatile purposes in TERM. Low cytotoxicity of PNPs, good biocompatibility, higher permeation and retention (EPR) effect, ability to deliver poorly soluble drugs and sustained release of them, retaining bioactivity of bioactive agents from enzymatic degradation for tissue engineering applications make PNPs as one of the fastest growing platform to overcome obstacles in TERM. Most of the new PNPs systems are designed to be sensitive to different physicochemical stimuli such as magnetic field, temperature, enzymes, pH, light, reducing/oxidizing agents which helps delivery or targeting systems with high specificity and efficiency for TERM applications , self-assembly, supercritical fluid (SCF) technology, nanoprecipitation, salting-out, dialysis, and microfluidic systems . Better regeneration compared to SDF-1α alone (control group) has been obtained as demonstrated by histological analysis, mRNA and protein levels for collagen type II, SOX9 and aggrecan.Recently, plasma protein based NPs have gained attention due to their high bioavailability, non-toxicity, biodegradability, ease of manipulation, long Fibrin as another plasma protein has been used for encapsulation of VEGF for promoting the angiogenesis for wound healing applications  or growth factors  are incorporated in them  PLLA/tetrahydrofuran through polymerization/thermal induced phase separation (TIPS) technique to produce PLLA based nanofibrous construct with conductivity feature for bone TE  (PNIPAM) NPs have been developed for slow VEGF delivery within the collagen hydrogels  in the major organs of the mononuclear phagocyte system were comparable while they were ~2-fold higher than that of the PEG NPs. Similar systems can be applied for production of various NPs with similar structure but different type of materials and their biological response can be studied to select the most suitable candidate for various TERM applications.Surface features play a critical role in biocompatibility features of biomaterials and strength of host immune system reactions. In a study, polymer model based particles were manufactured from degradable mesoporous silica template and they were used to study the effect of various surface-cell interactions without substrate dependency , ultrasound, MRI, NIR fluorescence imaging are among the widely used imaging techniques which alone or in conjugation with each other can provide sensitive, high resolution images from tissues through utilization of contrast agents  based NPs which are made through cell surface targeting molecular templates enable the construction of highly specific binding, 3D structures with affinity similar to antibodies for cell and tissue imaging. By mimicking glucuronic acid which is present in the form of hyaluronan on the surface of cells such as keratinocytes, Kunath et al. have imprinted glucurconic acid using (N-acrylamido) benzamidine (AAB) and methacrylamide (MAM) and polymerizable derivative of rhodamine as dye label for the detection of glycosylations on cell surface  fluorogen of benzothiadiazole-based emissive aggregate, PITBT-TPE, NPs labeled with cell penetrating Tat peptides (AIE-Tat NPs) for monitoring/tracking of bone marrow MSCs in HA scaffolds for monitoring bone regeneration in mouse  (PDLLA) diol to form two different PU NPs  have favorable mechanical properties, high biocompatibility, and tunable chemical structures which enable them to be used as 3D printing feeder for construction of custom made scaffolds. Hung et al. have used a synthetic biodegradable PU elastic NPs with hyaluronan (HA) as viscosity and cell aggregation enhancer, and Y27632 inhibitor as an alternative for transforming growth factor beta-3 (TGF-β3) for chondrogenic differentiation of MSCs as manufacture feeder materials (ink) for water based 3D printing for customized cartilage tissue engineering  dimethacrylate (PEGDMA) 3D printed scaffold containing bioactive glass (BG) 45S5 or HAp NPs for bone marrow derived human MSCs encapsulation  provide a strong toolbox for tissue engineered artificial organ functionalization.NV proposed the topic. MF-A, HK-M, CRdS, and JB wrote the initial and revised drafts. EB, AT, and NV provided feedback through the writing process and completed revisions based on feedback provided.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


There is increasing evidence to suggest that arctic cultures and ecosystems have followed non-linear responses to climate change. Norse Scandinavian farmers introduced agriculture to sub-arctic Greenland in the late tenth century, creating synanthropic landscapes and utilising seasonally abundant marine and terrestrial resources. Using a niche-construction framework and data from recent survey work, studies of diet, and regional-scale climate proxies we examine the potential mismatch between this imported agricultural niche and the constraints of the environment from the tenth to the fifteenth centuries. We argue that landscape modification conformed the Norse to a Scandinavian style of agriculture throughout settlement, structuring and limiting the efficacy of seasonal hunting strategies. Recent climate data provide evidence of sustained cooling from the mid thirteenth century and climate variation from the early fifteenth century. Archaeological evidence suggests that the Norse made incremental adjustments to the changing sub-arctic environment, but were limited by cultural adaptations made in past environments. Construet al.Niche construction (NC) can be defined as behaviours whereby organisms, or agents, modify environments in such a way that ‘selective pressures’ acting on organisms are changed  time delays between climate change and the response of vegetation communities  took place within a wider context of raiding, trading, and exploration known as the Viking Age (c. 793–1066 AD). Recent evidence suggests that the Norse settlers were seeking trade goods to replace ivory and hide lost following the extirpation of walrus in Iceland. In Greenland, walrus, fur bearing animals, and arctic exotica  were abundant , pigs, horses, dogs, and cats) characteristic of manor farms in contemporaneous Norway indicating the cultural significance of animal husbandry to Norse identity and status  grazing near settlement areas suggests that Norse settlers imported terrestrial hunting techniques developed in Scandinavia (McGovern Phoca groenlandicus) and hooded (Cystophora cristata) seals in the spring months   even early settlers would have experienced harsh winters and summer droughts in Greenland  declined significantly after AD 1300 likely due to sea-ice, displaced harbour seal populations, and increasing dependence on migratory seal species  in Norway and the Kalmar Union of 1397 AD effectively shifted the center of power from Norway to Denmark Epstein . Danish et al.These transitions on the European continent had contrasting impacts on North Atlantic trade. Unlike Iceland, Greenland lacked the combination of a laboring population, grazing sheep herds, and access to fish stocks to generate surpluses of wool and dried fish for export Madsen . An abseVinland Sagas and Ivar Bardarson’s accounts remain ambiguous and uncorroborated in the archaeological record , and the organisation of resources within a local catchment , Lake Vatnahverfi (c. AD 1290–1400), and Saqqaa Tasia (c. AD 1220–1380) of the Vatnahverfi region between the late-fourteenth and early-fifteenth centuries . The onset of cooler climatic conditions from the late-thirteenth century would have increased stress on homefield production, and subsequently stretched the application of hunting strategies. The construction of a farming niche would have distinguished between a socialised inside and a hostile outside (Arneborg Agricultural instability and climate uncertainty is a likely cause of social-ecological disequilibrium. The Norse made incremental changes to their diet, tools, buildings, clothing, and field systems without discernible impacts on their identity as Scandinavian-European farmers. These changes were sufficient to sustain subsistence in the mild conditions of the MCA (Arneborg . Cultura


Delayed graft function (DGF) increases the risk of graft loss by up to 40%, and recent developments in kidney donation have increased the risk of its occurrence. Lowering the risk of DGF, however, is challenging due to a complicated etiology in which ischemia-reperfusion injury (IRI) leads to acute tubular necrosis. Among various strategies explored, the choice of induction therapy is one consideration. Rabbit antithymocyte globulin (rATG [Thymoglobuline]) has complex immunomodulatory effects that are relevant to DGF. In addition to a rapid and profound T-cell depletion, rATG inhibits leukocyte migration and adhesion. Experimental studies of rATG have demonstrated attenuated IRI-related tissue damage in reperfused tissues, consistent with histological evidence from transplant recipients. Starting rATG intraoperatively instead of postoperatively can improve kidney graft function and reduce the incidence of DGF. rATG is effective in preventing acute rejection in kidney transplant recipients at high immunological risk, supporting delayed calcineurin inhibitor (CNI) introduction which protects the graft from early insults. A reduced rate of DGF has been reported with rATG (started intraoperatively) and delayed CNI therapy compared to IL-2RA induction with immediate CNI in patients at high immunological risk, but not in lower-risk patients. Overall, induction with rATG induction is the preferred choice for supporting delayed introduction of CNI therapy to avoid DGF in high-risk patients but shows no benefit versus IL-2RA in lower-risk individuals. Evidence is growing that intraoperative rATG ameliorates IRI, and it seems reasonable to routinely start rATG before reperfusion. Delayed graft function (DGF) remains a major barrier to expanding the donor pool for kidney transplantation and improving outcomes. It is estimated to affect between 23% and 38% of deceased-donor adult kidney transplant recipients –3, basedThe choice of induction therapy is one consideration. Rabbit antithymocyte globulin (rATG) is generally used preferentially in patients at high immunological risk, such as sensitized individuals, and in patients with other risk factors for DGF including older donor age and longer cold ischemia time , 17. As  et al. observed a threefold increase in DGF among recipients of a DCD graft in their analysis of Organ Procurement and Transplant (OPTN) data from 2003 to 2006 ). The higher doses of rATG which were used in the 1990s and early 2000s and which were associated with increased complications  have sin et al. . AnalyseDefining the contribution of a single intervention to the risk of DGF is difficult given the wide variations in donor management, graft quality, patients' risk status, donor quality, and immunosuppressive protocols. The current evidence base does not indicate a significant benefit for rATG induction in terms of preventing DGF in lower-risk patients; here, IL-2RA induction seems to be equally effective. However, there are now sufficient data to conclude that rATG induction—started intraoperatively—can help to avoid DGF in high-risk patients, reinforcing its benefit in the lowering of BPAR in these vulnerable individuals.


Treating diseases at their earliest stages significantly increases the chance of survival while decreasing the cost of treatment. Therefore, compared to traditional blood testing methods it is the goal of medical diagnostics to deliver a technique that can rapidly predict and if required non-invasively monitor illnesses such as lung cancer, diabetes, melanoma and breast cancer at their very earliest stages, when the chance of recovery is significantly higher. To date human breath analysis is a promising candidate for fulfilling this need. Here, we highlight the latest key achievements on nanostructured chemiresistive sensors for disease diagnosis by human breath with focus on the multi-scale engineering of both composition and nano-micro scale morphology. We critically assess and compare state-of-the-art devices with the intention to provide direction for the next generation of chemiresistive nanostructured sensors. A. A3, NO,In a similar approach, Kahn et al.  fabricatNon-invasive detection of diseases by analyzing human breath is a fast, low-cost and a simple alternative to blood analysis. Using semiconducting metal oxide gas sensors capable of analyzing human breath for medical applications have recently attracted great attention because of their high sensitivity, simple device fabrication, and great miniaturization possibility. Despite several challenges such as sensor’s selectivity, slow dynamic response and high operating temperature significant effort and careful investigation of different types of metal oxide semiconductor-based sensors including doped, composite and p-n heterojunction devices is proceeding with a focus on identifying the optimum nanostructure architecture and morphology. Regardless chemiresistive sensors based on metal oxide semiconductors are showing promising early results in the diagnostics of numerous diseases such as lung and breast cancers, asthma and diabetes. However, comprehensive work remains to be carried out regarding current and future technologies for diseases diagnosis using chemiresistive gas sensors.


Communication between cells is quintessential for biological function and cellular homeostasis. Membrane-bound extracellular vesicles known as exosomes play pivotal roles in mediating intercellular communication in tumor microenvironments. These vesicles and exosomes carry and transfer biomolecules such as proteins, lipids and nucleic acids. Here we focus on exosomes secreted from senescent cells. Cellular senescence can alter the microenvironment and influence neighbouring cells via the senescence-associated secretory phenotype (SASP), which consists of factors such as cytokines, chemokines, matrix proteases and growth factors. This review focuses on exosomes as emerging SASP components that can confer pro-tumorigenic effects in pre-malignant recipient cells. This is in addition to their role in carrying SASP factors. Transfer of such exosomal components may potentially lead to cell proliferation, inflammation and chromosomal instability, and consequently cancer initiation. Senescent cells are known to gather in various tissues with age; eliminating senescent cells or blocking the detrimental effects of the SASP has been shown to alleviate multiple age-related phenotypes. Hence, we speculate that a better understanding of the role of exosomes released from senescent cells in the context of cancer biology may have implications for elucidating mechanisms by which aging promotes cancer and other age-related diseases, and how therapeutic resistance is exacerbated with age. Cellular senescence is a cellular stress response that culminates in a state of stable cell cycle arrest . As suchThe main focus of this review aims to discuss the emerging cell non-autonomous role of senescence-derived exosomes and its possible implications for tumorigenesis. We first take a look at exosomal biogenesis and their functional roles upon uptake in premalignant and cancer cells. We then highlight the role of exosomes during senescence, with a key focus on exosomes as constituents of the senescent secretome known as senescence-associated secretory phenotype (SASP). Lastly, we provide an overall perspective as well as speculate on the implications of exosomes as pro-tumourigenic SASP in the “aging-cancer” nexus.Extracellular vesicles are membrane-bound vesicles released by multiple cell types that include immune cells, prostate epithelial cells, stem cells, cancer cells, and neurons . These iExosomes are basically nano-sized (ranging from 40–100 nm) intercellular communication shuttles. Since the discovery of exosomes in 1983, it has become evident that exosomes contribute to many aspects of physiology and disease, mainly via cell-to-cell communication . We highExosome biogenesis begins with inward budding of the cellular plasma membrane to form early endosomes. Inward budding of this endosomal membrane then forms late endosomes containing intraluminal vesicles . These eThe biogenesis of exosomes in MVBs can be delineated into two major pathways: (1) endosomal sorting complex required for transport (ESCRT)-dependent and (2) ESCRT-independent pathways ,13,14,15The molecular profiles of exosomes produced through their biogenesis resembles that of the parental cell and contain endocytic proteins ,22,23. TThe exosomal membrane consists of proteins related to the endosomal membrane transport and fusion pathway , and the ESCRT pathway  . In addiOnce exosomes are secreted, they travel through the extracellular millieu and get taken up by cells via (a) direct signalling through surface molecules, (b) fusion with target cell membrane, (c) receptor-dependent endocytosis and (d) the phagocytosis of opsonized exosomes  39]. Ex. Ex39]. Direct interaction between tumor cells and the tumor microenvironment (TME) is crucial for cancer progression . Many stIntercellular transfer of oncoproteins via exosomes has been proposed to facilitate tumor growth and metastasis. Peinado et al. demonstrated that highly metastatic melanoma cells secrete exosomes that increased the growth and metastasis of primary tumors, and programmed bone marrow progenitors towards a pro-angiogenic phenotype via receptor tyrosine kinase MET . In anotCancer-cell derived exosomes have also been found to contain factors that may enhance angiogenesis. Park et al. demonstrated by proteomic analysis that exosomes derived from hypoxic tumor cells contained abundant angiogenic proteins . AnotherExosomes are critical mediators of the communication between cancer cells and immune cells. While exosomes can function to activate immune response and exert anti-tumor activities ,59,60,61Tumor-derived exosomes also carry various mRNAs that have pro-tumorigenic functions. Skog et al. reported that exosomes generated from glioblastoma tumor cells contained mRNAs related to tumor progression, such as proliferation and immune evasion. These mRNAs were observed to be translated into functional protein when the exosomes were taken up by recipient cells, suggesting that glioblastoma tumor cells have the potential to transform their tumor microenvironment via secretion of these exosomes . In anotExcitingly, mRNA levels from exosomes may also serve as surrogates in monitoring disease response in the host. Muller et al. obtained exosomes purified from the pre/post vaccination plasma of patients with gliomas in an anti-tumor vaccine Phase I/II clinical trial and compared their mRNA expression levels of 24 immunoregulatory genes . They reMicroRNA (miRNA) profiles correlating to disease states have been detected in exosomes circulating in the host and have been found in saliva , blood pIn vitro and mouse studies have identified specific miRNAs in exosomes that were able to affect recipient cell states. A study by Zhou et al. observed that exosome-mediated transfer of miR-105 from metastatic breast cancer cells to endothelial cells affected the integrity of vascular barriers, contributing to metastasis . AnotherMammalian cells can enter cellular senescence, a state of stable cell cycle arrest, when faced with cellular stress and irreparable damage . AlthougIn addition to an intrinsic cell-autonomous tumor-suppressive mechanisms , SASP coVarious studies have shown the ability of SASP factors to establish immunosuppressive microenvironments, thereby promoting tumor growth ,97. For Recent evidence has demonstrated that exosomes constitute part of the SASP and can mediate paracrine signaling effects on the microenvironment ,100. IndOne of the first reports involving senescent cells revealed a significant increase in secretion of exosome-like vesicles in irradiation-induced senescent prostate cancer cells as well as in normal human diploid fibroblasts undergoing proliferative senescence . The stuNotably, senescent cells accumulate with age and exosomes secreted from senescent cells vary from that of younger cells . Indeed,Additionally, our unpublished data indicate that the impact of exosomes released from mitotic-slippage induced senescent (MIS)  breast cIn this review, we have demonstrated that exosomes play pivotal roles in intercellular communication, cellular homeostasis and disease. The discovery of exosomes secreted by malignant cells afforded new hypotheses in the field especially in the study of metastasis, senescence and therapy resistance. We believe that the bioactive molecules present in the tumour microenvironment described in This is exciting as it provides new avenues of inhibiting cancer progression by targeting tumour-derived exosomes or inhibiting exosome biogenesis. A deeper understanding of exosome biogenesis, loading and internalization of specific cargo within senescent cells, and their subsequent uptake in recipient cells could also shed light on mechanisms behind enhanced secretion of exosomes in senescent cells. Such a study could also provide novel targets to pursue in cancer therapy. Additionally, exosomes by themselves could potentially be used as a therapeutic to deliver drugs and biomolecules  to alter cellular function in recipient cells and serve as attractive candidates for next-generation targeted drug delivery . For exaThe field, however, does face several challenges, especially in the areas of exosomal research methodologies and tools in vivo. Apart from studies on exosomes as biomarkers, the majority of exosome research in cancer has been performed in vitro. Due to the mixture of subtypes of circulating exosomes and cells that are isolated from bodily fluids, advances in exosome isolation, quantification and analysis techniques would be helpful to the study of their pathological roles in vivo. Additionally, to observe their uptake and track exosomes in animal models, in vivo studies often use exosomes extracted from in vitro cell culture. Hence, future studies focused on better elucidation of the function and influence of exosomes in the tumor microenvironment in vivo will benefit the field immensely.


Neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are increasing in prevalence but lack targeted therapeutics. Although the pathological mechanisms behind these diseases remain unclear, both ALS and FTD are characterized pathologically by aberrant protein aggregation and inclusion formation within neurons, which correlates with neurodegeneration. Notably, aggregation of several key proteins, including TAR DNA binding protein of 43 kDa (TDP-43), superoxide dismutase 1 (SOD1), and tau, have been implicated in these diseases. Proteomics methods are being increasingly applied to better understand disease-related mechanisms and to identify biomarkers of disease, using model systems as well as human samples. Proteomics-based approaches offer unbiased, high-throughput, and quantitative results with numerous applications for investigating proteins of interest. Here, we review recent advances in the understanding of ALS and FTD pathophysiology obtained using proteomics approaches, and we assess technical and experimental limitations. We compare findings from various mass spectrometry (MS) approaches including quantitative proteomics methods such as stable isotope labeling by amino acids in cell culture (SILAC) and tandem mass tagging (TMT) to approaches such as label-free quantitation (LFQ) and sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH-MS) in studies of ALS and FTD. Similarly, we describe disease-related protein-protein interaction (PPI) studies using approaches including immunoprecipitation mass spectrometry (IP-MS) and proximity-dependent biotin identification (BioID) and discuss future application of new techniques including proximity-dependent ascorbic acid peroxidase labeling (APEX), and biotinylation by antibody recognition (BAR). Furthermore, we explore the use of MS to detect post-translational modifications (PTMs), such as ubiquitination and phosphorylation, of disease-relevant proteins in ALS and FTD. We also discuss upstream technologies that enable enrichment of proteins of interest, highlighting the contributions of new techniques to isolate disease-relevant protein inclusions including flow cytometric analysis of inclusions and trafficking (FloIT). These recently developed approaches, as well as related advances yet to be applied to studies of these neurodegenerative diseases, offer numerous opportunities for discovery of potential therapeutic targets and biomarkers for ALS and FTD. Neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), demonstrate dysfunctional protein clearance and accumulation of protein-rich inclusions in neuronal cells. Resolving whether these inclusions are a cause of cellular degeneration or a symptom of homeostatic dysfunction has proven difficult, and the pathological mechanisms underlying their formation are still largely unknown. However, through attempting to answer these questions, the fundamental roles of protein aggregation and protein clearance in the pathology of these diseases have been established. Recent advances in proteomics technologies have advanced our understanding of both the primary pathological proteins as well as other associated proteins that may play downstream roles in disease mechanisms. The purpose of this review is to evaluate the current proteomic tools and techniques being used to understand these diseases, to summarize the state of knowledge gained from proteomic studies on ALS and FTD, and to discuss development of more effective disease-modifying treatments and biomarkers for clinical assessment driven by new advances in proteomic technologies.ALS and FTD are both debilitating neurodegenerative diseases caused by selective loss of subsets of neurons. In ALS, disease pathology primarily affects the motor neurons of the primary motor cortex and spinal cord , and in C9ORF72, are also characterized by the presence of TDP-43 pathology   further for peptide identification. The data generated by MS can be searched using algorithms that incorporate protein databases to generate lists of identified proteins. The two major streams for quantitative proteomics that are widely used are label-free and labeled quantitation  technology in the past decade, with higher resolution and faster scan rates, has enabled smoother and quicker identification of highly complex proteomes with shorter analysis periods . The typC-MS/MS) . The peptitation . Both meLabel-free quantitation (LFQ) can use either data-dependent or data-independent acquisition (DIA) analyses. Data-dependent acquisition quantitation can be performed on either spectral counts or spectrometric signal intensity of product ions from selected precursors generated by the mass spectrometer . RecentlG93A mice over three pre-symptomatic time points in disease , which cause rare familial cases of ALS/FTD, have also been studied by overexpression in neuronal cell lines and zebrafish to investigate the effects and mechanisms of ALS/FTD  are missed in precursor ion selection. In order to improve this coverage, additional sample prefractionation steps (such as strong cation exchange or basic reverse phase chromatography) can be used to reduce the complexity of the sample to be analyzed by MS. A recent LFQ approach that is being more widely adopted relies on data independent quantitation with methods such as Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH) that attempt to circumvent some of these issues associated with analyzing complex samples by data-dependent acquisition.SWATH, or data-independent acquisition (DIA), acquires data by cycling through predefined sequential windows of a chromatographic elution range generating a larger number of identified proteins from a complex mixture . RecentlMatrix-assisted laser desorption ionization (MALDI) is an alternative method to introduce a sample into a MS. This differs from electro-spray ionization (ESI) through focusing laser energy at a matrix-embedded sample for low fragmentation and reduced multi-charged ion states . TypicalLabeled-based approaches have considerably higher quantitation accuracy in exchange for lower proteome coverage compared to LFQ . The labStable Isotope Labeling by Amino acids in Cell culture (SILAC) labeling is achieved by growing cells in culture with growth medium containing different isotopically labeled amino acids that are incorporated during protein synthesis . The proThe versatility of SILAC can extend to the generation of an internal standard in a cell-line to quantify low abundance proteins in tissue (known as “Super SILAC”). This was applied to investigate the accumulated proteins in detergent-insoluble brain lysates from four control and four FTLD brain samples, with comparison to a HEK293 standard . A summaUnlike SILAC, which incorporates isotopically labeled amino acids at the protein level, the Tandem Mass Tag (TMT) system is used to label peptides following proteolytic digestions . TMT tagG93A, TMT-labeling of peptides combined with the results of RNA-Seq demonstrated impairments to nuclear and cytoplasmic transport  using protein A or G conjugated to beads . Of noteBioID and APEX are proximity-ligation methods which facilitate the covalent attachment of biotin onto proteins in close proximity to a protein of interest. The application of these methods for interrogating PPIs of proteins is based on the strong interaction between biotin and streptavidin  and the ∗) is also able to convert biotin to a highly reactive intermediate, however, due to this R118G mutation, the intermediate is prematurely released from the active site of BirA∗ and diffuses away leading to biotinylation of nearby lysine residues  and biotin phenol is added to media such that APEX can catalyze a one-electron oxidization of biotin phenol to a biotinphenoxyl radical which subsequently biotinylates proteins in proximity  , thereby2O2 . APEX ca2O2 .Drosophila ALS models, suggesting disturbances in SGs may be related to disease pathogenesis  is another proximity-labeling technique which has recently been established for investigating PPIs in fixed samples . Here, cPost-translational modifications provide the basis of biological diversity in the proteome by enabling the same protein to carry out diverse functions through characteristic variations of modifications and their temporal regulation. PTMs are small, covalent, amino acid modifications added onto a protein; they are highly dynamic and play key roles in selectively regulating protein function in cells, tissue, and biofluids, such as CSF . PTMs rePathological phosphorylation of proteins such as tau, TDP-43, α-synuclein or FUS contribute to the formation of protein inclusions in neurodegenerative disease . For exaMany PTMs can be mapped by tandem MS (MS/MS) , which oA popular enrichment strategy for identifying ubiquitination sites on peptides called diglycine (diGLY) enrichment exploits the cleavage sites (K-𝜀-GG) of ubiquitinated proteins following trypsin digestion . The cov2), immobilized metal affinity chromatography (IMAC), and hydrophilic interaction chromatography (HILIC), discussed elsewhere (3PO4 (-m/z 98) in the mass spectra. Many algorithms have been developed for interpreting MS/MS fragmentation spectra and phosphorylation site localization such as Mascot Delta Score  could also help to establish common pathways involved in this process across different types of neurodegenerative diseases.Fluorescence activated cell sorting (FACS) is one technique that can be used to purify insoluble protein or inclusions from cell culture or tissues. Regarding cell-based models of neurodegenerative disease-associated protein aggregation, gentle plasma membrane permeabilization in combination with FACS could be employed to purify inclusions for proteomic analyses. A method for this was recently described, whereby cells are permeabilized with 0.5% (v/v) TritonX-100 in PBS supplemented with protease/phosphatase inhibitors to quantify and characterize inclusions comprised of fluorescently tagged proteins of interest by flow cytometry . This woPurification of inclusions by FACS for proteomic analysis has previously been adopted to investigate the protein composition of Huntington’s disease-associated polyglutamine inclusions , 2006. UAs high-throughput technologies have advanced, many proteomic-based biomarker studies have presented an abundance of potential candidates; however, it is often not feasible to properly validate each individual protein considering the heterogeneity amongst different patient cohorts, which can hinder the level of accuracy and reproducibility. Selection of which candidates to validate is not clearly defined, uniform, or standardized. This selection can be made based on a combination of statistics, current literature as well as assessed potential based on PPIs, both predicted and real.One of the major issues that arises as a result of subjective validation is the variation of proteins identified across parallel studies, rarely reinforcing the previously presented data. For example, measurements of HeLa cells prepared identically and analyzed across different laboratories using the same instrumentation revealed a high level of biological heterogeneity on both transcriptomic and proteomic levels . This ocIn recent years, an increased number of small and large-scale biomarker studies have been performed on human ALS samples by LFQ, as recently reviewed . CSF froBiofluid sample usage is prominent due to the need for a directly translatable and easily retrievable marker for rapid and accurate diagnosis. Alongside disease diagnosis, the ability to distinguish between subtypes of a disease would prove instrumental in developing personalized treatments. Whilst many studies have identified altered proteins across cohorts of patients, others focus on teasing apart unclear differences such as those found in subtypes of FTD . RecentlThe technologies presented in this review have demonstrated their utility when applied to neurodegenerative diseases such as ALS and FTD. However, there needs to be a focus on validating the research laterally, solidifying findings to progress with ensuing biological questions. Incorporation of parallel approaches such as transcriptomics using RNASeq alongside results from proteomics can reinforce potential hits of interest. Studies have successfully combined transcriptomic and proteomics findings to provide mechanistic insights, such as the finding of lysosomal changes in Grn-/- mice that are modulated by the FTD-risk protein TMEM106B . AdditioEach technique discussed in this review has advantages and limitations, described above and in additional detail elsewhere . ImportaIn this review we have identified proteomics papers relevant to ALS/FTD and presented them alongside discussion of current technologies. The consistency amongst proteins identified strictly depend on the disease type/model used, however, in ALS research, TDP-43 as well as hnRNPs appear to reoccur . An asseIn the context of ALS and FTD, greater focus should be given to comparisons between new and existing proteomics datasets as the growing number of studies are released using a variety of available techniques. Further comparisons between ALS and FTD datasets and those obtained from other neurodegenerative diseases should also be performed, since there are likely to be overlap in mechanisms which may be broadly involved in neurodegeneration. This may also provide information on how disease-specific alterations arise and why certain cell populations or CNS regions are susceptible to different neurodegenerative disease processes. For example, recent studies have applied TMT to identify changes in various Alzheimer’s disease mice, identifying hundreds of differentially expressed proteins . PreviouFinally, it will also be informative to use these proteomic techniques to more closely investigate biochemical changes longitudinally in disease, particularly in studies of model systems from which brain and spinal cord samples can be collected at pre-defined time points. Strategies such as this are most likely to reveal the earliest pathogenic changes in disease, which may be most amenable to therapeutic intervention, but which may not be identified by studies at later disease time points or by using end-stage human tissue samples alone. The use of comparative studies and recent advances in new technologies and labeling techniques offers great hope for future understanding of ALS and FTD, and development of clinical tests and therapeutics based on these findings.TH, AW, and AL conceived of the manuscript and drafted the tables. All authors were responsible for drafting, writing and editing, and read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.