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Background and aims: OxLDL modulates innate and adaptive immunity, and extracellular vesicles (EVs) released from both non-immune and immune cells are proposed key players in atherosclerosis development. In the present study, we aimed to investigate EVs expressing markers related to adaptive immunity-driven inflammation and endothelial activation/dysfunction in hypercholesterolemic patients. Methods: EVs were phenotyped in thirty patients with familial hypercholesterolemia (FH) and twenty-three healthy controls using the Extracellular Vesicle (EV) Array with antibodies targeting proteins expressed on B and T cells, and endothelial cells. Results: FH patients had a higher atherosclerotic burden, as determined by the mean carotid intima-media thickness (IMT) (0.64 ± 0.12 mm vs. 0.58 ± 0.07 mm; p = 0.033), higher oxLDL levels (p

Background: Blood–barrier (BBB) breakdown and active inflammation are hallmarks of relapsing multiple sclerosis (RMS), but the molecular events contributing to the development of new lesions are not well explored. Leaky endothelial junctions are associated with increased production of endothelial-derived extracellular microvesicles (EVs) and result in the entry of circulating immune cells into the brain. MRI with intravenous gadolinium (Gd) can visualize acute blood–barrier disruption as the initial event of the evolution of new lesions. Methods: Here, weekly MRI with Gd was combined with proteomics, multiplex immunoassay, and endothelial stress-optimized EV array to identify early markers related to BBB disruption. Five patients with RMS with no disease-modifying treatment were monitored weekly using high-resolution 3T MRI scanning with intravenous gadolinium (Gd) for 8 weeks. Patients were then divided into three groups (low, medium, or high MRI activity) defined by the number of new, total, and maximally enhancing Gd-enhancing lesions and the number of new FLAIR lesions. Plasma samples taken at each MRI were analyzed for protein biomarkers of inflammation by quantitative proteomics, and cytokines using multiplex immunoassays. EVs were characterized with an optimized endothelial stress EV array based on exosome surface protein markers for the detection of soluble secreted EVs. Results: Proteomics analysis of plasma yielded quantitative information on 208 proteins at each patient time point (n = 40). We observed the highest number of unique dysregulated proteins (DEPs) and the highest functional enrichment in the low vs. high MRI activity comparison. Complement activation and complement/coagulation cascade were also strongly overrepresented in the low vs. high MRI activity comparison. Activation of the alternative complement pathway, pathways of blood coagulation, extracellular matrix organization, and the regulation of TLR and IGF transport were unique for the low vs. high MRI activity comparison as well, with these pathways being overrepresented in the patient with high MRI activity. Principal component analysis indicated the individuality of plasma profiles in patients. IL-17 was upregulated at all time points during 8 weeks in patients with high vs. low MRI activity. Hierarchical clustering of soluble markers in the plasma indicated that all four MRI outcomes clustered together with IL-17, IL-12p70, and IL-1β. MRI outcomes also showed clustering with EV markers CD62E/P, MIC A/B, ICAM-1, and CD42A. The combined cluster of these cytokines, EV markers, and MRI outcomes clustered also with IL-12p40 and IL-7. All four MRI outcomes correlated positively with levels of IL-17 (p < 0.001, respectively), and EV-ICAM-1 (p < 0.0003, respectively). IL-1β levels positively correlated with the number of new Gd-enhancing lesions (p < 0.01), new FLAIR lesions (p < 0.001), and total number of Gd-enhancing lesions (p < 0.05). IL-6 levels positively correlated with the number of new FLAIR lesions (p < 0.05). Random Forests and linear mixed models identified IL-17, CCL17/TARC, CCL3/MIP-1α, and TNF-α as composite biomarkers predicting new lesion evolution. Conclusions: Combination of serial frequent MRI with proteome, neuroinflammation markers, and protein array data of EVs enabled assessment of temporal changes in inflammation and endothelial dysfunction in RMS related to the evolution of new and enhancing lesions. Particularly, the Th17 pathway and IL-1β clustered and correlated with new lesions and Gd enhancement, indicating their importance in BBB disruption and initiating acute brain inflammation in MS. In addition to the Th17 pathway, abundant protein changes between MRI activity groups suggested the role of EVs and the coagulation system along with innate immune responses including acute phase proteins, complement components, and neutrophil degranulation.

Abstract Plasma extracellular vesicle (EV) number and composition are altered following myocardial infarction (MI), but to properly understand the significance of these changes it is essential to appreciate how the different isolation methods affect EV characteristics, proteome and sphingolipidome. Here, we compared plasma EV isolated from platelet‐poor plasma from four healthy donors and six MI patients at presentation and 1‐month post‐MI using ultracentrifugation (UC), polyethylene glycol precipitation, acoustic trapping, size‐exclusion chromatography (SEC) and immunoaffinity capture. The isolated EV were evaluated by Nanoparticle Tracking Analysis (NTA), Western blot, transmission electron microscopy (TEM), an EV‐protein array, untargeted proteomics (LC‐MS/MS) and targeted sphingolipidomics (LC‐MS/MS). The application of the five different plasma EV isolation methods in patients presenting with MI showed that the choice of plasma EV isolation method influenced the ability to distinguish elevations in plasma EV concentration following MI, enrichment of EV‐cargo (EV‐proteins and sphingolipidomics) and associations with the size of the infarct determined by cardiac magnetic resonance imaging 6 months post‐MI. Despite the selection bias imposed by each method, a core of EV‐associated proteins and lipids was detectable using all approaches. However, this study highlights how each isolation method comes with its own idiosyncrasies and makes the comparison of data acquired by different techniques in clinical studies problematic.

Extracellular vesicles (EVs) have become a topic of interest within the field of diagnostic biomarkers; however, recent developments in the study of EVs have increased the need for simpler but still comprehensive methods for characterization. Here, we describe how to simultaneously measure several surface or surface-associated proteins on EVs using a multiparametric microarray-based analysis termed Extracellular Vesicle Array (EV Array), which is developed to catch and phenotypically characterize small EVs. Previously, this analysis has been limited to measuring only one fluorescent signal per analysis. The analysis relies on antibodies printed onto a solid surface, for catching the EVs carrying the specific surface or surface-associated proteins, and on the subsequent fluorescent detection. For the optimization of detection, two antibodies with attached Cy3 or Cy5 were added to various combinations of the EV surface or surface-associated proteins: CD9, CD63, CD81, flotillin-1, and HSP90. In this study, the EV surface or surface-associated proteins were analyzed in human plasma from six healthy subjects. Changes observed in signal intensities from Cy3 and Cy5 related specifically to these combinations and allowed for a comparison of the two different fluorescent signals. When comparing the results, it was observed that it is possible to measure the EV surface or surface-associated proteins at both 532 nm (Cy3) and 635 nm (Cy5) simultaneously without a significant change in signals from the detection molecules. This allows us to measure multiple EV marker proteins in a single analysis, thereby more quickly finding complex biomarker patterns in a sample.

Noise from wind turbines (WTs) has been reported more annoying than traffic noise at similar levels, and concerns have been raised about whether WT noise (WTN) can increase risk for cardiovascular disease. We aimed to investigate if long-term exposure to WTN increases risk for hypertension, estimated as redemption of prescriptions for antihypertensive drugs. We identified all Danish dwellings within a radius of 20 WT heights from a WT and 25% randomly selected dwellings within 20–40 WT heights radius. Using data on WT type and hourly wind conditions at each WT, we estimated hourly outdoor (10–10,000 Hz) and low frequency (LF: 10–160 Hz) indoor WTN for all dwellings, and aggregated it as long-term nighttime running means. From nationwide registries, we identified 535,675 persons age 25–85 years living in these dwellings for >1 year from 1996 to 2013, of whom 83,729 fulfilled our case definition of redeeming ≥2 prescriptions and ≥180 defined daily doses of antihypertensive drugs within a year. Data were analyzed using Poisson regression according to categories of WTN exposure and adjustment for individual and area-level covariates. We found no associations between 5-year mean exposure to WTN during night and redemption of antihypertensives, with hazard ratios (HR) of 0.91 (95% confidence intervals (CI): 0.78–1.06) for outdoor WTN ≥ 42 dB(A) and of 1.06 (CI: 0.83–1.35) for indoor LF WTN ≥ 15 dB(A) when compared to the reference WTN levels (

Introduction: High arsenic concentration in drinking water is associated with a higher incidence rate of stroke, but only few studies have investigated an association with arsenic in drinking water at low concentration (

Physical activity initiates a wide range of multi-systemic adaptations that promote mental and physical health. Recent work demonstrated that exercise triggers the release of extracellular vesicles (EVs) into the circulation, possibly contributing to exercise-associated adaptive systemic signalling. Circulating EVs comprise a heterogeneous collection of different EV-subclasses released from various cell types. So far, a comprehensive picture of the parental and target cell types, EV-subpopulation diversity and functional properties of EVs released during exercise (ExerVs) is lacking. Here, we performed a detailed EV-phenotyping analysis to explore the cellular origin and potential subtypes of ExerVs. Healthy male athletes were subjected to an incremental cycling test until exhaustion and blood was drawn before, during, and immediately after the test. Analysis of total blood plasma by EV Array suggested endothelial and leukocyte characteristics of ExerVs. We further purified ExerVs from plasma by size exclusion chromatography as well as CD9-, CD63- or CD81-immunobead isolation to examine ExerV-subclass dynamics. EV-marker analysis demonstrated increasing EV-levels during cycling exercise, with highest levels at peak exercise in all EV-subclasses analysed. Phenotyping of ExerVs using a multiplexed flow-cytometry platform revealed a pattern of cell surface markers associated with ExerVs and identified lymphocytes (CD4, CD8), monocytes (CD14), platelets (CD41, CD42, CD62P), endothelial cells (CD105, CD146) and antigen presenting cells (MHC-II) as ExerV-parental cells. We conclude that multiple cell types associated with the circulatory system contribute to a pool of heterogeneous ExerVs, which may be involved in exercise-related signalling mechanisms and tissue crosstalk.

With the expansion of robotic applications in the industrial domain, it is important that the robots can execute their tasks in a safe and reliable way. A monitoring system can be implemented to ensure the detection of abnormal situations of the robots and report the abnormality to their human supervisors or cooperators. In this work, we focus on developing a modeling framework for monitoring robotic system based on means-end analysis and the concept of action phases from action theory. A circular cascaded action phase structure is proposed for building the model of cyclical robotic events. This functional model provide a formal way of decompose robotic tasks and analyze each level of conditions for an action to be executed successfully. It can be used for monitoring robotic systems by checking the preconditions in the action phases and identifying the failure modes. The proposed method is demonstrated by using a simulated robotic manipulation system. The simulation results demonstrate the feasibility of the developed functional model in finding errors during the execution monitoring.

Abstract Cells release lipid-bound extracellular vesicles (EVs; exosomes, microvesicles and apoptotic bodies) containing proteins, lipids and RNAs into the circulation. Vesicles mediate intercellular communication between both neighboring and distant cells. There is substantial interest in using EVs as biomarkers for age-related diseases including cancer, and neurodegenerative, metabolic and cardiovascular diseases. The majority of research focuses on identifying differences in EVs when comparing disease states and matched controls. Here, we analyzed circulating plasma EVs in a cross-sectional and longitudinal study in order to address age-related changes in community-dwelling individuals. We found that EV concentration decreases with advancing age. Furthermore, EVs from older individuals were more readily internalized by B cells and increased MHC-II expression on monocytes compared with EVs from younger individuals, indicating that the decreased concentration of EVs with age may be due in part to increased internalization. EVs activated both monocytes and B cells, and activation of B cells by LPS enhanced EV internalization. We also report a relative stability of EV concentration and protein amount in individual subjects over time. Our data provide important information towards establishing a profile of EVs with human age, which will further aid in the development of EV-based diagnostics for aging and age-related diseases.

Aims/hypothesis: It has been suggested that air pollution may increase the risk of type 2 diabetes but data on particulate matter with diameter

Tissue factor (TF)–bearing microvesicles (MVs) and exosomes may play a role in hemostasis and thrombosis. MVs may be quantified by flow cytometry (FC)–based detection of phosphatidylserine (PS)-positive submicron particles carrying specific antigens, although interference from lipoproteins complicates this approach. In this study, we evaluated the effect of food intake on blood levels of TF-bearing particles measured by FC and small extracellular vesicles (EVs) measured by a protein microarray–based test termed EV Array. Platelet-free plasma (PFP) was obtained from 20 healthy persons in the fasting state and 75 minutes after consumption of a meal. Postprandial changes in the concentration of PS-positive particles, including subgroups binding labeled antibodies against TF, CD41, CD146, and CD62E, respectively (FC), small EVs (EV Array), and TF antigen and procoagulant phospholipids (PPLs) were measured. Furthermore, we tested the effect on FC results of in vitro addition of lipoproteins to fasting PFP. We found significantly increased plasma concentrations of PS-positive particles and all examined subgroups postprandially, while no changes in small EVs, PPL, or TF antigen levels were found. Levels of all types of particles measured by FC were also elevated by lipoprotein spiking. In conclusion, meal consumption as well as in vitro addition of lipoproteins to fasting plasma induces increased levels of PS-positive particles as measured by FC, including TF-positive subtypes and subtypes exposing other antigens. While the observed postprandial increase may to some extent reflect elevated MV levels, our results indicate a substantial interference from lipoproteins.

Introduction: Nanoparticle tracking analysis (NTA) enables measurement of extracellular vesicles (EVs) but lacks the ability to distinct between EVs and lipoproteins which are abundantly present in blood plasma. Limitations in ultracentrifugation and size exclusion chromatography applied for EV isolation may result in inadequate EV purification and preservation. In this proof of concept study, we aimed to evaluate the potential of antibody-mediated removal of lipoproteins from plasma prior to extracellular vesicle (EV) analysis by nanoparticle tracking analysis (NTA). Methods: Ten platelet-free plasma (PFP) samples from healthy fasting subjects were incubated with magnetic beads coated with antibodies against apolipoprotein B-48 and B-100 (ApoB). Plasma samples were analysed with NTA before and after application of the bead procedure. Four fasting PFP samples were analysed with an ELISA specific for human ApoB to estimate the degree of removal of lipoproteins and EV array analysis was used for identification of possible EV loss. Results: The magnetic bead separation procedure resulted in a median reduction of the particle concentration in plasma by 62% (interquartile range 32–72%). The mean size of the remaining particles generally increased. ApoB concentration was reduced to a level close to the background signal, whereas a median reduction of the EV content by 21% (range 8–43%) was observed. Conclusion: Anti-ApoB antibody coated magnetic beads may hold potential for removal of lipoproteins from human PFP prior to EV measurement by NTA but some artefactual effect and EV loss may have to be endured.

Extracellular vesicles (EVs) and exosomes are difficult to enrich or purify from biofluids, hence quantification and phenotyping of these are tedious and inaccurate. The multiplexed, highly sensitive and high-throughput platform of the EV Array presented by Jørgensen et al., (J Extracell Vesicles, 2013; 2: 10) has been refined regarding the capabilities of the method for characterization and molecular profiling of EV surface markers. Here, we present an extended microarray platform to detect and phenotype plasma-derived EVs (optimized for exosomes) for up to 60 antigens without any enrichment or purification prior to analysis.

Background: Lung cancer is one of the leading causes of cancer-related death. At the time of diagnosis, more than half of the patients will have disseminated disease and, yet, diagnosing can be challenging. New methods are desired to improve the diagnostic work-up. Exosomes are cell-derived vesicles displaying various proteins on their membrane surfaces. In addition, they are readily available in blood samples where they constitute potential biomarkers of human diseases, such as cancer. Here, we examine the potential of distinguishing non-small cell lung carcinoma (NSCLC) patients from control subjects based on the differential display of exosomal protein markers. Methods: Plasma was isolated from 109 NSCLC patients with advanced stage (IIIa–IV) disease and 110 matched control subjects initially suspected of having cancer, but diagnosed to be cancer free. The Extracellular Vesicle Array (EV Array) was used to phenotype exosomes directly from the plasma samples. The array contained 37 antibodies targeting lung cancer-related proteins and was used to capture exosomes, which were visualised with a cocktail of biotin-conjugated CD9, CD63 and CD81 antibodies. Results: The EV Array analysis was capable of detecting and phenotyping exosomes in all samples from only 10 µL of unpurified plasma. Multivariate analysis using the Random Forests method produced a combined 30-marker model separating the two patient groups with an area under the curve of 0.83, CI: 0.77–0.90. The 30-marker model has a sensitivity of 0.75 and a specificity of 0.76, and it classifies patients with 75.3% accuracy. Conclusion: The EV Array technique is a simple, minimal-invasive tool with potential to identify lung cancer patients.

Extracellular vesicles (EVs) are involved in inter-cellular communication and their cargo may provide prognostic/diagnostic biomarkers. To discover EV-associated biomarkers for Multiple Sclerosis (MS), we used an immune marker array to identify surface proteins on circulating EVs that differ between MS patients and controls (n = 3 each). We identified toll-like receptor-3 (TLR3) as a potential target for further validation. We utilized prospectively collected serum from relapsing-remitting MS patients (n = 18) and controls (n = 16) and confirmed lower concentration of TLR3 and higher concentration of mechanistically related TLR4 in MS EVs compared to controls. Future studies may further evaluate the utility of EV-associated TLRs as MS biomarkers and uncover their mechanistic significance.

Background: Exosomes are one of the several types of cell-derived vesicles with a diameter of 30–100 nm. These extracellular vesicles are recognized as potential markers of human diseases such as cancer. However, their use in diagnostic tests requires an objective and high-throughput method to define their phenotype and determine their concentration in biological fluids. To identify circulating as well as cell culture-derived vesicles, the current standard is immunoblotting or a flow cytometrical analysis for specific proteins, both of which requires large amounts of purified vesicles. Methods: Based on the technology of protein microarray, we hereby present a highly sensitive Extracellular Vesicle (EV) Array capable of detecting and phenotyping exosomes and other extracellular vesicles from unpurified starting material in a high-throughput manner. To only detect the exosomes captured on the EV Array, a cocktail of antibodies against the tetraspanins CD9, CD63 and CD81 was used. These antibodies were selected to ensure that all exosomes captured are detected, and concomitantly excluding the detection of other types of microvesicles. Results: The limit of detection (LOD) was determined on exosomes derived from the colon cancer cell line LS180. It clarified that supernatant from only approximately 104 cells was needed to obtain signals or that only 2.5×104 exosomes were required for each microarray spot (~1 nL). Phenotyping was performed on plasma (1–10 µL) from 7 healthy donors, which were applied to the EV Array with a panel of antibodies against 21 different cellular surface antigens and cancer antigens. For each donor, there was considerable heterogeneity in the expression levels of individual markers. The protein profiles of the exosomes (defined as positive for CD9, CD63 and CD81) revealed that only the expression level of CD9 and CD81 was approximately equal in the 7 donors. This implies questioning the use of CD63 as a standard exosomal marker since the expression level of this tetraspanin was considerably lower.

Appropriate and well-documented in vitro cell-culturing systems are necessary to study the activity and biological function of extracellular vesicles (EVs). The aim of this study was to describe an experimental system, in which dynamic, vesicle-based cell communication can be investigated. A commercially available cell-culturing system was applied to study contact-independent cell communication, which separated two cell populations using a membrane with a pore size of 0.4 μm. The EV exchange characteristics between the two compartments in the culture set-up was preliminarily investigated in a cell-free set-up, and analysed using the Extracellular Vesicle (EV) Array and Nanoparticle Tracking Analysis. The application of the cell-culturing set-up was demonstrated using co-cultures of human primary cells. The effects of the relative placement of the two cell populations on the phenotype of EVs found in the cell supernatant were investigated. The results indicate that this placement can be important for the biological hypothesis that is being investigated. These observations are relevant for short (

Extracellular vesicles (EVs) have a demonstrated involvement in modulating the immune system. It has been proposed that EVs could be used as biomarkers for detection of inflammatory and immunological disorders. Consequently, it is of great interest to investigate EVs in more detail with focus on immunological markers. In this study, five major leukocyte subpopulations and the corresponding leukocyte-derived EVs were phenotyped with focus on selected immunological lineage-specific markers and selected vesicle-related markers. The leukocyte-derived EVs displayed phenotypic differences in the 34 markers investigated. The majority of the lineage-specific markers used for identification of the parent cell types could not be detected on EVs released from monocultures of the associated cell types. In contrast, the vesicular presentation of CD9, CD63, and CD81 correlated to the cell surface expression of these markers, however, with few exceptions. Furthermore, the cellular expression of CD9, CD63, and CD81 varied between leukocytes present in whole blood and cultured leukocytes. In summary, these data demonstrate that the cellular and vesicular presentation of selected lineage-specific and vesicle-related markers may differ, supporting the accumulating observations that sorting of molecular cargo into EVs is tightly controlled.

Extracellular vesicles (EVs) are one of several tools that cells use to communicate with each other. This communication is facilitated by a number of surface-associated proteins and the cargo of the vesicles. For several cancer types, the amount of EVs is observed to be up-regulated in patients compared to healthy individuals, possibly signifying the presence of an aberrant process. The hypoxia-induced release of EVs from cancer cells has been hypothesized to cause the malignant transformation of healthy recipient cells. In this study, the phenotype of cells and EVs from the ovarian cancer cell lines, COV504, SKOV3, and Pt4, were quantified and analysed under normoxic and hypoxic conditions. It was shown that both cells and EVs express common markers and that the EV phenotype varies more than the cellular phenotype. Additionally, cells subjected to 24 hours of hypoxia compared to normoxia produced more EVs. The phenotyping of EVs from cancer cell lines provides information about their molecular composition. This information may be translated to knowledge regarding the functionality of EVs and lead to a better understanding of their role in cancer.

Non-Hodgkin lymphoma (NHL) is a frequent cancer and incidence rates have increased markedly during the second half of the 20(th) century; however, the few established risk factors cannot explain this rise and still little is known about the aetiology of NHL. Spatial analyses have been applied in an attempt to identify environmental risk factors, but most studies do not take human mobility into account. The aim of this study was to identify clustering of NHL in space and time in Denmark, using 33 years of residential addresses. We utilised the nation-wide Danish registers and unique personal identification number that all Danish citizens have to conduct a register-based case-control study of 3210 NHL cases and two independent control groups of 3210 each. Cases were identified in the Danish Cancer Registry and controls were matched by age and sex and randomly selected from the Civil Registration System. Residential addresses of cases and controls from 1971 to 2003 were collected from the Civil Registration System and geocoded. Data on pervious hospital diagnoses and operations were obtained from the National Patient Register. We applied the methods of the newly developed Q-statistics to identify space-time clustering of NHL. All analyses were conducted with each of the two control groups, and we adjusted for previous history of autoimmune disease, HIV/AIDS or organ transplantation. Some areas with statistically significant clustering were identified; however, results were not consistent across the two control groups; thus we interpret the results as chance findings. We found no evidence for clustering of NHL in space and time using 33 years of residential histories, suggesting that if the rise in incidence of NHL is a result of risk factors that vary across space and time, the spatio-temporal variation of such factors in Denmark is too small to be detected with the applied method.

BackgroundThe enzyme indoleamine 2,3-dioxygenase (IDO) exerts an well established immunosuppressive function in cancer. IDO is expressed within the tumor itself as well as in antigen-presenting cells in tumor-draining lymph nodes, where it promotes the establishment of peripheral immune tolerance to tumor antigens. In the present study, we tested the notion whether IDO itself may be subject to immune responses.Methods and findingsThe presence of naturally occurring IDO-specific CD8 T cells in cancer patients was determined by MHC/peptide stainings as well as ELISPOT. Antigen specific cytotoxic T lymphocytes (CTL) from the peripheral blood of cancer patients were cloned and expanded. The functional capacity of the established CTL clones was examined by chrome release assays. The study unveiled spontaneous cytotoxic T-cell reactivity against IDO in peripheral blood as well as in the tumor microenvironment of different cancer patients. We demonstrate that these IDO reactive T cells are indeed peptide specific, cytotoxic effector cells. Hence, IDO reactive T cells are able to recognize and kill tumor cells including directly isolated AML blasts as well as IDO-expressing dendritic cells, i.e. one of the major immune suppressive cell populations.ConclusionIDO may serve as an important and widely applicable target for anti-cancer immunotherapeutic strategies. Furthermore, as emerging evidence suggests that IDO constitutes a significant counter-regulatory mechanism induced by pro-inflammatory signals, IDO-based immunotherapy holds the promise to boost anti-cancer immunotherapy in general.

Aim: Vote counting is frequently used in meta-analyses to rank biomarker candidates, but to our knowledge, there have been no independent assessments of its validity. Here, we used predictions from a recent meta-analysis to determine how well number of supporting studies, combined sample size and mean fold change performed as vote-counting strategy criteria., Materials & Methods: Fifty miRNAs previously ranked for their ability to distinguish lung cancer tissue from normal were assayed by RT-qPCR using 45 paired tumor-normal samples., Results: Number of supporting studies predicted biomarker performance (p = 0.0006; r = 0.44), but sample size and fold change did not (p > 0.2)., Conclusion: Despite limitations, counting the number supporting studies appears to be an effective criterion for ranking biomarkers. Predictions based on sample size and fold change provided little added value. External validation studies should be conducted to establish the performance characteristics of strategies used to rank biomarkers.

Dietary restriction (DR), one of the most robust life-extending manipulations, is usually associated with reduced adiposity. This reduction is hypothesized to be important in the life-extending effect of DR, because excess adiposity is associated with metabolic and age-related disease. Previously, we described remarkable variation in the lifespan response of 41 recombinant inbred strains of mice to DR, ranging from life extension to life shortening. Here, we used this variation to determine the relationship of lifespan modulation under DR to fat loss. Across strains, DR life extension correlated inversely with fat reduction, measured at midlife (males, r= -0.41, P<0.05, n=38 strains; females, r= -0.63, P<0.001, n=33 strains) and later ages. Thus, strains with the least reduction in fat were more likely to show life extension, and those with the greatest reduction were more likely to have shortened lifespan. We identified two significant quantitative trait loci (QTLs) affecting fat mass under DR in males but none for lifespan, precluding the confirmation of these loci as coordinate modulators of adiposity and longevity. Our data also provide evidence for a QTL previously shown to affect fuel efficiency under DR. In summary, the data do not support an important role for fat reduction in life extension by DR. They suggest instead that factors associated with maintaining adiposity are important for survival and life extension under DR., (© 2011 The Authors. Aging Cell © 2011 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland.)

Aging leads to a loss of thermoregulation that can be readily monitored in laboratory mice. However, it is unclear from previous studies-we provide a tabular summary of 15 articles-whether significant loss occurs by midlife ( approximately 15 months of age). In this study, we examined 34 females from 22 LSXSS strains starting at 4 and 8 months of age (17 mice per age group). We used transponders inserted just under the loose skin of the pelt and calibrated against rectal body temperature to measure temperatures quickly without restraint. We found that the mean body temperatures measured 5 months later (9 and 13 months of age) had dropped significantly below normal in both groups: 0.6 masculineC lower in the younger cohort and 1.0 masculineC lower in the older cohort. These drops were not associated with weight loss or signs of pathology. Notably, the loss of thermoregulation between 8 and 13 months of age also exhibited genetic variation that was highly significant (P = 0.004). Such variation is potentially a powerful tool for determining the cause of thermoregulatory loss with age and whether this loss predicts senescence changes later in life, including the force of mortality.

Chronic dietary restriction (DR) is considered among the most robust life-extending interventions, but several reports indicate that DR does not always extend and may even shorten lifespan in some genotypes. An unbiased genetic screen of the lifespan response to DR has been lacking. Here, we measured the effect of one commonly used level of DR (40% reduction in food intake) on mean lifespan of virgin males and females in 41 recombinant inbred strains of mice. Mean strain-specific lifespan varied two to threefold under ad libitum (AL) feeding and 6- to 10-fold under DR, in males and females respectively. Notably, DR shortened lifespan in more strains than those in which it lengthened life. Food intake and female fertility varied markedly among strains under AL feeding, but neither predicted DR survival: therefore, strains in which DR shortened lifespan did not have low food intake or poor reproductive potential. Finally, strain-specific lifespans under DR and AL feeding were not correlated, indicating that the genetic determinants of lifespan under these two conditions differ. These results demonstrate that the lifespan response to a single level of DR exhibits wide variation amenable to genetic analysis. They also show that DR can shorten lifespan in inbred mice. Although strains with shortened lifespan under 40% DR may not respond negatively under less stringent DR, the results raise the possibility that life extension by DR may not be universal.

Numerous physiological and molecular changes accompany dietary restriction (DR), which has been a major impediment to elucidating the causal basis underlying DR's many health benefits. Two major metabolic responses to DR that potentially underlie many of these changes are the body temperature (T(b)) and body weight (BW) responses. These responses also represent an especially difficult challenge to uncouple during DR. We demonstrate in this study, using two recombinant inbred (RI) panels of mice (the LXS and LSXSS) that naturally occurring genetic variation serves as a powerful tool for modulating T(b) and BW independently during DR. The correlation coefficient between the two responses was essentially zero, with R = -0.04 in the LXS and -0.03 in the LSXSS, the latter averaged across replicate cohorts. This study is also the first to report that there is highly significant (P = 10(-10)) strain variation in the T(b) response to DR in the LXS (51 strains tested), with strain means ranging from 2 to 4 degrees C below normal. The results suggest that the strain variation in the T(b) response to DR is largely due to differences in the rate of heat loss rather than heat production (i.e., metabolic rate). This variation can thus be used to assess the long-term effects of lower T(b) independent of BW or metabolic rate, as well as independent of food intake and motor activity as previously shown. These results also suggest that murine genetic variation may be useful for uncoupling many more responses to DR.

We present genetic analyses of murine weight loss during dietary restriction (DR) for females eating 60% ad libitum (AL). We examined 5 cohorts across 81 different strains (22 strains tested twice) that included the LXS and LSXSS recombinant inbred strains, the LXS parental strains ILS and ISS, and the classical inbreds 129S6, A, BALB/c, C57BL/6, C3H, and DBA. Weight loss exhibited highly significant genetic variation, with DR body weights ranging from approximately 60 to approximately 85% of AL body weight. This variation was not explained by the strain differences in absolute food intake, feces calorie content, motor activity, or AL body fat. Heritability was 40-50%, and several provisional quantitative trait loci were mapped. This variation can be used to test whether weight loss correlates with the health benefits of DR, independently of the reduction in calories. The genetic variation also implies the existence of genes that would be novel therapeutic targets, distinct from genes affecting AL body weight or body fat, for enhancing (or mitigating) weight loss during food restriction.

Establishing the mechanism of action of general anesthetics at the molecular level is difficult because of the multiple targets with which these drugs are associated. Inbred short sleep (ISS) and long sleep (ILS) mice are differentially sensitive in response to ethanol and other sedative hypnotics and contain a single quantitative trait locus (Lorp1) that accounts for the genetic variance of loss-of-righting reflex in response to propofol (LORP). In this study, we used high-density oligonucleotide microarrays to identify global gene expression and candidate genes differentially expressed within the Lorp1 region that may give insight into the molecular mechanism underlying LORP. Microarray analysis was performed using Affymetrix MG-U74Av2 Genechips and a selection of differentially expressed genes was confirmed by semiquantitative reverse transcription-polymerase chain reaction. Global expression in the brains of ILS and ISS mice revealed 3423 genes that were significantly expressed, of which 139 (4%) were differentially expressed. Analysis of genes located within the Lorp1 region showed that 26 genes were significantly expressed and that just 2 genes (7%) were differentially expressed. These genes encoded for the proteins AWP1 (associated with protein kinase 1) and "BTB (POZ) domain containing 1," whose functions are largely uncharacterized. Genes differentially expressed outside Lorp1 included seven genes with previously characterized neuronal functions and thus stand out as additional candidate genes that may be involved in mediating the neurosensitivity differences between ISS and ILS.

Modification of any one of three transmembrane protein tyrosine kinase (PTK) genes, old-1, old-2 (formerly tkr-1 and tkr-2, respectively), and daf-2 can extend the mean and maximum life span of the nematode Caenorhabditis elegans. To identify paralogs and orthologs, we delineated relationships between these three PTKs and all known transmembrane PTKs and all known mammalian nontransmembrane PTKs using molecular phylogenetics. The tree includes a number of invertebrate receptor PTKs and a novel mammalian receptor PTK (inferred from the expressed-sequence tag database) that have not previously been analyzed. old-1 and old-2 were found to be members of a surprisingly large C. elegans PTK family having 16 members. Interestingly, only four members of this transmembrane family appeared to have receptor domains (immunoglobulin-like in each case). The C-terminal domain of this family was found to have a unique sequence motif that could be important for downstream signaling. Among mammalian PTKs, the old-1/old-2 family appeared to be most closely related to the Pdgfr, Fgfr, Ret, and Tie/Tek families. However, these families appeared to have split too early from the old-1/old-2 family to be orthologs, suggesting that a mammalian ortholog could yet be discovered. An extensive search of the expressed-sequence tag database suggested no additional candidate orthologs. In contrast to old-1 and old-2, daf-2 had no C. elegans paralogs. Although daf-2 was most closely related to the mammalian insulin receptor family, a hydra insulin receptor-like sequence suggested that daf-2 might not be an ortholog of the insulin receptor family. Among PTKs, the old-1/old-2 family and daf-2 were not particularly closely related, raising the possibility that other PTK families might extend life span. On a more general note, our survey of the expressed-sequence tag database suggested that few, if any, additional mammalian PTK families are likely to be discovered. The one novel family that was discovered could represent a novel oncogene family, given the prevalence of oncogenes among PTKs. Finally, the PTK tree was consistent with nematodes and fruit flies being as divergent as nematodes and mammals, suggesting that life extension mechanisms shared by nematodes and fruit flies would be reasonable candidates for extending mammalian life spans.

Background: Long-sleep (LS) and short-sleep (SS) mice, initially selected for differential sensitivity to ethanol, also exhibit differential sensitivity to propofol. By interbreeding LS and SS mice to obtain progeny whose chromosomes are a patchwork of the LS and SS chromosomes, the authors determined whether differential propofol sensitivity cosegregates with any particular chromosomal region(s). Such cosegregation is the essence of genetic linkage mapping and a first step toward isolating a gene that can modulate propofol sensitivity in mammals. A gene underlying a quantitative trait such as anesthetic sensitivity is commonly called a quantitative trait locus (QTL)., Methods: The propofol dose was 20 mg/kg injected retroorbitally. Sensitivity was measured as the duration of the loss of righting reflex (LORR). The LORR and propofol brain levels at awakening were determined for 24 LSXSS recombinant-inbred (RI) strains, derived by intercrossing LS and SS for two generations followed by >20 generations of inbreeding. A genetic linkage between LORR and an albino mutation on chromosome 7 was investigated further using 164 second-generation progeny (F2s) from intercrossing inbred LS and inbred SS mice, similar to the LSXSS RIs except F2s are not inbred. The linkage between propofol sensitivity and the albino locus also was investigated using additional genetic markers on chromosome 7. Statistical significance was assessed by interval mapping using a regression method for RIs and Mapmaker/QTL (Whitehead Institute, Cambridge, MA) for F2s., Results: Genetic mapping in the LSXSS RIs revealed a QTL tightly linked to the Tyr (albino) locus that accounts for nearly all of the genetic difference in propofol sensitivity between LS and SS mice. Analysis of propofol brain levels at awakening indicated that this QTL results from differential neurosensitivity. Mapping in F2s confirmed the genetic linkage to Tyr. Mice (ISS c/c x C57BL/6 c2j/C) that differed only by an albino mutation at Tyr were not differentially sensitive to propofol., Conclusions: A single QTL, called Lorp1, underlies most of the genetic difference in propofol neurosensitivity between LS and SS mice. Although this QTL is tightly linked to Tyr, propofol sensitivity is not modulated by albinism. For mapping this QTL, the LSXSS RIs proved to be an especially powerful resource, localizing the candidate-gene region to a 99% confidence interval of only 2.5 centimorgans.

The murine albino-deletion complex developed as part of the Oak Ridge specific-locus test covers 6-11 cM of chromosome 7. This complex has proven to be a valuable resource for localizing traits to a small target region for positional cloning. In this study, we mapped the endpoints of deletions in this complex using all of the available Mit simple-sequence length polymorphism (SSLP) markers. Concurrently, this mapping has determined the map order of nearly all of the SSLP markers, most of which were previously unresolved. The SSLP-based deletion map was confirmed and genetic distances were determined using the European Collaborative Interspecific Backcross panel of nearly a thousand mice. The average SSLP marker resolution is 0.3-0.4 cM, comparable to the cloning capacity of yeast artificial chromosomes (YACs). The SSLP markers were then used to construct a genetically anchored YAC framework map that further confirms the deletion map. We find that the largest deleted region distal to Tyr is about two to three times larger than the largest proximal deletion region, and the original C3H/101 regions flanking the deletions (moved to an St2A cch/cch background) are smaller than anticipated, which we suggest may result from increased recombination rates immediately flanking the deleted regions.

The inbred mouse strain, C57BL/6J, was derived from mice of the Mus musculus complex. C57BL/6J can be crossed in the laboratory with a closely related mouse species, M. spretus to produce fertile offspring; however there has been no previous evidence of gene flow between M. spretus and M. musculus in nature. Analysis of the repetitive sequence LINE-1, using both direct sequence analysis and genomic Southern blot hybridization to species-specific LINE-1 hybridization probes, demonstrates the presence of LINE-1 elements in C57BL/6J that were derived from the species M. spretus. These spretus-like LINE-1 elements in C57BL/6J reveal a cross to M. spretus somewhere in the history of C57BL/6J. It is unclear if the spretus-like LINE-1 elements are still embedded in flanking DNA derived from M. spretus or if they have transposed to new sites. The number of spretus-like elements detected suggests a maximum of 6.5% of the C57BL/6J genome may be derived from M. spretus.

LINE-1 is the major family of long, interspersed, repetitive DNA sequences found in mammalian genomes. The mouse species Mus spretus contains large LINE-1 subfamilies that are distinguishable from the LINE-1 elements of laboratory Mus domesticus strains by their content of particular nucleotide differences. Oligonucleotides containing these differences act as M. spretus-specific LINE-1 hybridization probes. We have used these probes as a novel genetic tool in conjunction with an interspecific hybrid congenic mouse, in which the M. spretus allele of the pearl gene has been transferred onto a M. domesticus background. From a lambda library prepared from this congenic mouse, four clones were isolated by hybridization to the M. spretus-specific probes. After derivation of genetic markers from these clones, two of them were found to be linked to the pearl gene. These markers are the first two of up to 75 that could be isolated to support cloning the pearl gene. Considering the interspersed nature of LINE-1, we propose that species-specific LINE-1 probes could also be used to isolate markers for many other target genes.

Mus domesticus and Mus spretus mice are closely related subspecies. For genetic investigations involving hybrid mice, we have developed a set of species-specific oligonucleotide probes based on the detection of LINE-1 sequence differences. LINE-1 is a repetitive DNA family whose many members are interspersed among the genes. In this study, library screening experiments were used to fully characterize the species specificity of four M. domesticus LINE-1 probes and three M. spretus LINE-1 probes. It was found that the nucleotide differences detected by the probes define large, species-specific subfamilies. We show that collaborative use of such probes can be employed to selectively detect thousands of species-specific library clones. Consequently, these probes could be exploited to monitor and access almost any given species-specific region of interest within hybrid genomes.