Genetic risk models could potentially be useful in identifying high-risk groups for the prevention of complex diseases. We investigated the performance of this risk stratification strategy by examining epidemiological parameters that impact the predictive ability of risk models.We assessed sensitivity, specificity, and positive and negative predictive value for all possible risk thresholds that can define high-risk groups and investigated how these measures depend on the frequency of disease in the population, the frequency of the high-risk group, and the discriminative accuracy of the risk model, as assessed by the area under the receiver-operating characteristic curve (AUC). In a simulation study, we modeled genetic risk scores of 50 genes with equal odds ratios and genotype frequencies, and varied the odds ratios and the disease frequency across scenarios. We also performed a simulation of age-related macular degeneration risk prediction based on published odds ratios and frequencies for six genetic risk variants.We show that when the frequency of the high-risk group was lower than the disease frequency, positive predictive value increased with the AUC but sensitivity remained low. When the frequency of the high-risk group was higher than the disease frequency, sensitivity was high but positive predictive value remained low. When both frequencies were equal, both positive predictive value and sensitivity increased with increasing AUC, but higher AUC was needed to maximize both measures.The performance of risk stratification is strongly determined by the frequency of the high-risk group relative to the frequency of disease in the population. The identification of high-risk groups with appreciable combinations of sensitivity and positive predictive value requires higher AUC. There is increasing interest in the potential use of testing multiple genetic variants for the prediction of common complex diseases such as type 2 diabetes, osteoporosis and cardiovascular disease, particularly because this could help targeting preventive and therapeutic interventions to individuals and groups with high genetic risk. While to date most genetic risk models show only modest predictive performance -7, improDespite the modest predictive ability, some argue that genetic risk models can still be useful in health care and disease prevention to identify individuals at very high risk . PreventFor single genetic tests, the relationship between the epidemiological assessment of the genetic association ) and the predictive accuracy of the test  have been described by simple arithmetic formulas . These fIn this study, we examined the performance of risk stratification based on genetic risk models that include multiple variants simultaneously. We investigated sensitivity, specificity, PPV and NPV of genetic risk models along the range of threshold values that can be chosen to define high-risk groups. This detailed exploration of the interrelationships between sensitivity, PPV, prevalence of risk group and disease prevalence using genetic risk scores instead of single risk variants has not been reported before. We repeated the analyses for thresholds that define high-risk groups with a frequency lower, equal or higher than the disease frequency for increasing values of the area under the receiver operating characteristic curve (AUC). To address these objectives we used simulated data across a wide variety of ORs and frequencies for genetic variants. We also carried out an additional simulation based on published ORs and frequencies for six genetic polymorphisms predicting age-related macular degeneration (AMD) risk .For the construction of simulated data sets, we used a modeling procedure that has been described in detail elsewhere . In shorCFH , LOC387715 (rs10490924), C2 (rs9332739), CFB (rs641153) and C3 (rs2230199). For each locus we considered the effect from the univariate logistic regression analysis with AMD as outcome variable and the genetic variants as predictor variables. For each locus the three genotypes were entered independently, with the exception of C2 and CFB for which the genotypes were grouped in two categories, one conferring an increased risk of disease. Additional file We constructed a dataset using the disease risk from prevalence estimates in adults 40 years of age or over , and genIn the main simulation study, we constructed a genetic risk score that was a simple count of the number of risk genotypes. Note that this score has perfect correlation with predicted risk because all variants have the same frequency of the risk genotype and the same OR. The disease risk increases with the number of risk genotypes in the genetic risk model. In the AMD simulation, we derived predicted risks using logistic regression analysis with genetic risk variants entered as categorical variables. High-risk groups were defined as all individuals with risk scores above a chosen threshold.First, to evaluate the impact of genotype frequencies and ORs on the overall discriminative accuracy of genetic risk models, we assessed the AUC . Next, tFigure Figure Figure Increasing the OR of all variants included in the genetic risk score also increases the AUC of the risk score. Figure Finally, we examined the same associations using simulated data based on published ORs and frequencies for six known AMD genetic risk factors. The range of predicted risks was 0.2% to 62% (Additional file This study investigated the relationships between sensitivity, PPV, prevalence of risk group and disease prevalence when genetic risk scores, as opposed to single risk variants, are used for risk stratification. A major finding from this analysis is that when the frequency of the high-risk group approximates the disease frequency, both sensitivity and PPV increase with higher AUC. At all other frequencies of the high-risk group, higher AUC will increase either sensitivity or PPV. Selecting the optimal cut-off threshold will consequently be a trade-off between higher sensitivity at the price of lower PPV, or vice versa.While the relationship between the number of individuals carrying a certain genetic risk factor and the risk of disease in the population was shown to influence the screening performance for a single marker , we haveet al. [Genetic tests are usually assessed in terms of their ability to distinguish risk groups with large differences in risk. Nevertheless, it has been shown that large relative risks are not sufficient to demonstrate the model's clinical validity and utility -22. Measet al. . A higheThe observation that the sensitivity and PPV are equal when the frequency of the high-risk group equals the frequency of disease in the population holds across different settings. First, this relationship holds irrespective of whether the disease risk refers to the lifetime risk, a cumulative incidence over certain time period or the disease prevalence. Evidently, if we consider, for example, lifetime risks instead of 10-year risks, the frequency of the high-risk group for which the sensitivity and PPV are equal will be larger, because lifetime risks by definition are higher than 10-year risks. Then for the same AUC values, these larger high-risk groups will have higher sensitivity and PPV. However, prediction models that consider longer time periods generally have lower AUC, implying that combinations of higher sensitivity and PPV may not be observed. Put differently, lifetime risk models with lower AUC may yield the same sensitivity/PPV combination as 10-year risk models with higher AUC, but the value of using a model with low AUC may become questionable.Second, the relationship also holds irrespective of how the risks are calculated. There are several ways in which genetic risks can be expressed. One is to use a simple genetic risk score based on the number of risk alleles carried. This approach, which we used in our analyses, assumes that each allele has the same effect on the risk of disease ,25. AnotThird, the relationship also holds for risk models in general, that is, including other non-genetic risk models, such as the Framingham risk score for prediction of cardiovascular disease. Basically the relationship is valid for any continuous variable that is dichotomized to create risk groups, such as blood pressure, cholesterol or triglyceride level. This is also true for risk models that include together novel biomarkers and established risk factors, a topic that has recently attracted a lot of research ,27.When risk models are used to target interventions to high-risk subgroups, these subgroups are defined by choosing cut-off values for the predicted risks. The cut-off corresponding to a frequency of the high-risk group equal to the disease frequency optimizes both the sensitivity and the PPV, but is not necessarily optimal. Cut-off values are chosen on the basis of cost-benefit analyses, balancing the harms and benefits of false positive and false negative classifications of risk. The cut-off defining a risk group with a frequency equal to disease frequency is optimal only when the harm and benefit have equal weights. Selection of optimal cut-off based on a decision-analytic approach is a complex process that requires detailed input information of measures like sensitivity, specificity, PPV, NPV and related costs. For example, a recent study reported the effect of family history and 14 SNPs on the cost-effectiveness of chemoprevention with finasteride for prostate cancer . The resOur observations have implications for health care applications of genetic testing, but also for the direct-to-consumer offer of personal genome tests via the internet. For health care applications that need high PPV, such as targeting invasive interventions to people at the highest risk, a low AUC means that only a small proportion of this group will be identified. For applications that need high sensitivity, such as screening programs, the interventions will be given to a very large part of the population, mostly to people who will not develop the disease. And finally, low AUC means for personal genome testing that most people who will develop the disease will not be identified as having high risks.Anticipating the advances in this field, it is essential to develop more rigorous approaches to evaluate the clinical usefulness of risk models ,30. We hAMD: age-related macular degeneration; AUC: area under the receiver operating characteristic curve; NPV: negative predictive value; OR: odds ratio; PPV: positive predictive value; SNP: single-nucleotide polymorphism.The authors declare that they have no competing interests.ACJWJ and RM conceived the study and drafted the manuscript. RM performed the statistical analysis. RM and MJK participated in the design and helped to draft the manuscript. All authors read and approved the final manuscript.Supplementary tables and supplementary figure legends. A table listing genotype ORs and genotypic frequencies of the markers included in the AMD simulation and figure legends for Additional files Click here for fileSupplementary Figure S1. A figure showing the change in specificity and NPV for different thresholds.Click here for fileSupplementary Figure S2. A figure showing the sensitivity, specificity, PPV and NPV for different frequencies of the population at high risk.Click here for fileSupplementary Figure S3. A file showing the distribution of predicted risks in individuals with and without AMD.Click here for file


Currently it is uncertain how to define osteoporosis and who to treat after a hip fracture. There is little to support the universal treatment of all such patients but how to select those most in need of treatment is not clear. In this study we have compared cortical and trabecular bone status between patients with spinal fractures and those with hip fracture with or without spinal fracture with the aim to begin to identify, by a simple clinical method (spine x-ray), a group of hip fracture patients likely to be more responsive to treatment with current antiresorptive agents.Comparison of convenience samples of three groups of 50 patients, one with spinal fractures, one with a hip fracture, and one with both. Measurements consist of bone mineral density at the lumbar spine, at the four standard hip sites, number, distribution and severity of spinal fractures by the method of Genant, cortical bone thickness at the infero-medial femoral neck site, femoral neck and axis length and femoral neck width.Patients with spinal fractures alone have the most deficient bones at both trabecular and cortical sites: those with hip fracture and no spinal fractures the best at trabecular bone and most cortical bone sites: and those with both hip and spinal fractures intermediate in most measurements. Hip axis length and neck width did not differ between groups.The presence of the spinal fracture indicates poor trabecular bone status in hip fracture patients. Hip fracture patients without spinal fractures have a bone mass similar to the reference range for their age and gender. Poor trabecular bone in hip fracture patients may point to a category of patient more likely to benefit from therapy and may be indicated by the presence of spinal fractures. In the evaluation of medications for osteoporosis the essential requirement is the prevention of fractures. Although initially the focus was on the prevention of compression fractures of the spine, the importance of preventing non-spinal fractures has emerged and is now a critical expectation of the drugs. Without exception, however, the therapies are better at preventing spinal fractures than non-spinal fractures, possibly because they are more effective on trabecular bone than cortical bone, the latter being an important contributor to the strength of the non-spinal fracture sites . DespiteBy and large, it seems that patients at the lower end of the normal distribution of bone densities in the general population, are, should they fall, at greater risk of fracture than those at the upper end of the distribution. For example, in the EPIDOS study of women average age of 80 years, those with BMD on heel ultrasound and neck of femur BMD that were below average had a risk of hip fracture of 1.96% per year compared to 0.27% if above average .In addition to simple bone thinning with age, some architectural features of the neck of femur might be important in determining risk of hip fracture. Differences in hip bone density, mostly at the neck of femur site, and differences in architecture (Singh index) and geometry  of the proximal femur between those with a hip fracture and normal controls have been described -9. An adBy contrast, spinal fractures do not usually require a fall and can be sustained in normal activity. Indeed, the vertebral fracture is to some extent distinguished from the non-vertebral fracture in that it usually occurs under a force the vertebra should have been able to withstand, in contrast to the non-vertebral fracture where more is expected of the bone than it was designed to meet. Nonetheless, these non-vertebral fractures have come to be considered fragility fractures, and have come to be considered indicative of osteoporosis, although they are best predicted by a measure of cortical bone. Nonetheless, most of the clinical trials, predominantly with bisphosphonates, were done on people many or most of whom had spinal compression fractures and better fitted the trabecular bone loss model. These patients displayed anything up to 13% chance of a new spinal fracture within a year, higher if there had been a recent compression fracture, but a lesser degree of increased risk of non-vertebral fracture . Thus itThe question arises, when does the spinal fracture syndrome merge into or overlap with the mechanical model of low trauma fracture? Is there, at one end of the scale, the patient with a hip fracture who has deficient trabecular bone while at the other end the patient with perhaps some loss of cortical bone but in whom the fracture is mostly traumatic and where response to treatment is likely to be minimal? In other words, does the degree of trabecular bone deficiency indicate a state more likely to respond to treatment and how can these patients be identified? If, for example, hip fracture patients have compression fractures, is this on the basis of low trabecular bone mass, or are these also largely traumatic? If the former, this may point to a state of deficient trabecular bone mass throughout the skeleton, but if largely traumatic or mechanical in nature, bone mass may not be much reduced but we might expect a different distribution of spinal fractures, more being low in the lumbar spine, the site most vulnerable in a fall or accident .To begin to explore this possibility we have, in this study, compared, on the basis of trabecular and cortical measurements, three approximately age-matched groups of older patients with osteoporotic-type fractures consisting of; patients attending an out-patient osteoporosis clinic referred because of the presence of compression fractures (but with no history of hip fracture); patients admitted to a hip fracture rehabilitation program who, upon spinal x-ray, were found to have compression fractures; and similar patients found not to have compression fractures. The first group is proposed as the gold standard for the presence of severe trabecular bone loss, the second, hip and spinal fracture patients as potentially having trabecular osteoporosis  and the third, those with only a hip fracture, as potentially non-osteoporotic traumatic fracture patients.This was a retrospective data review of patients mostly over the age of 70 years presenting to an osteoporosis clinic with spinal compression fractures but no history of hip fracture and those admitted to a rehabilitation unit following a hip fracture. All patients had a standard osteoporosis series of radiographs performed, consisting of a lateral and AP view of the thoracic and lumbar spine and AP view of the pelvis, all in digital form. All patients underwent bone mineral density at spine and hip sites. Hip fracture patients from long term care, and those who are admitted directly to LTC from the orthopedic unit, are not transferred to the rehabilitation program and are not represented in this study. These patients are older than the population studied here. Likewise, younger patients who can be discharged directly home following surgery do not come to the rehabilitation program and are also not represented here.For this study there are a total of 150 patients comprising 50 patients from each of three patient groups, viz. patients from the osteoporosis clinic with compression fractures and no history of hip fracture, patients from the hip fracture rehabilitation population who were found to have spinal compression fractures on x-ray, and patients with a hip fracture but no spinal fracture. Patients were required to have had: (1) BMD; (2) radiographic images of the thoracic and lumbar spine; and (3) radiographic image of the pelvis with at least one clean hip joint (no prosthetic or replacement device).Several variables were examined as follows:The BMD for the spine (L2-L4), the total hip, the intertrochanteric region, the trochanteric and the neck of the proximal femur were recorded. All BMDs were obtained using dual-energy x-ray absorptiometry (DEXA). For each region tested the BMD value (g/cm2), t-score and z-score were recorded. The z-score is the degree in standard deviation units by which the individual differs from the mean level for their age and sex as compared to the normative data base for the specific BMD machine. We use this comparison to determine if the individuals and groups are similar or otherwise to the normal population. The spine site is predominantly a measure of trabecular bone (66%), the neck of femur site predominantly cortical bone (75%) while the intertrochanteric site is about 50% trabecular and 50% cortical . In the Radiographic images of the lateral vertebral column were recorded, graded, and analyzed based on Genant et al.’s semiquantitative assessment of vertebral fractures . This meThe Spinal Fracture Index used Genant’s semiquantitative assessment for each vertebrae from T4-L4 . The radAs spinal BMD is influenced by the presence of spinal degenerative disease, we recorded this when it was provided by the formal radiology report which was done by radiologists unaware of this study.Hip axis length, femoral neck length and femoral neck cortical bone thickness were measured as outlined by Peacock and colleagues . BrieflyU test was used to compare groups rather than the t-test used for the others.Statistical analysis was performed using IBMSPSS Statistics Software version 19. The SPSS Test of Normality employed was the Shapiro-Wilk Test which indicated normality for all parameters except trochanteric BMD, femoral neck BMD, femoral neck width and hip axis length for which the Mann–Whitney Ethics approval was obtained through the University of Western Ontario Ethics Board.Table Patients with only spinal fractures had a significantly lower lumbar t-score than those presenting with hip fractures  (−2.54 vs. -1.27 p<0.001). Twenty-three, 16 and 23 spinal fracture only patients, hip and spinal fracture patients and hip fracture only patients respectively had degenerative changes noted on the x-ray report and 9, 8, and 7 of these respectively were reported as showing sclerosis, but the spinal BMD was not higher in these patients and their exclusion did not affect the results. Spinal fracture only patients also have a tendency to a lower hip density, reaching significance only for the intertrochanteric (50% trabecular bone) region  and for men and women separately (r=0.41 for men and 0.45 for women p<0.023 and P<0.001 respectively).Neither the hip axis length, femoral neck length nor femoral neck width showed any difference between groups Table . FemoralWhen the BMD for the femoral neck is plotted on the CAROC graphs (3) of femoral neck t-score against age approximately one quarter of patients were of an age that was beyond the graphs’ limits. However, we attempted to extrapolate the graphs beyond the 85 year age limit, and found only a minority of women with hip fracture (approximately one third) and one out of every 20 men fell, on the basis of BMD alone, into the high risk category. Interestingly, the presence of spinal fractures did not increase the proportion of hip fracture patients falling into the high risk category when defined by the femoral neck t-score. For those with spinal fracture alone, only 14  fell into the high risk zone on the CAROC graph. On the other hand, of those with spinal fractures alone, 50% fell below a spinal t-score of −2.5, while 30% of those with hip and spinal fracture did so, and only 18% of those with a hip fracture alone. For a femoral neck t-score of −2.5, the respective numbers are 60%, 43% and 45% respectively.Patients presenting with spinal fractures have a higher number of spinal fractures than those with hip fracture and spinal fractures (165 vs. 138 respectively) and as a result both the spinal deformity index and the spinal fracture index are higher in the vertebral fracture alone group  but the individual fractures are of similar severity (1.728 vs. 1.66 ns.). However, as shown in Figure This study compared three groups of fracture patients: those with spinal fractures and no hip fractures, those with both spinal and hip fractures and those with hip fractures and no spinal fractures. Our analysis has attempted to sort out the relative contributions of trabecular and cortical bone deficiency to the different fractures and we were particularly interested to see if the presence of vertebral fractures in the ihip fracture population might identify a group with deficient trabecular bone and so be a group who could be responsive to treatment, as opposed to being a group in whom the presence of vertebral fractures was simply another reflection of their falling and hence more traumatic in nature. Patients presenting to the osteoporosis clinic with spinal fractures have generally more deficient bones than those of similar age presenting with hip fractures, including a tendency to a lower hip density. In most measurements the spinal fracture only patients have the lowest bones mass and the hip fracture patients without spinal fractures the highest and those with both spinal and hip fracture in between. This is clear for the spinal density but a similar, though less marked, trend is seen for the hip density measurements that reflect trabecular bone, and less again for the cortical site of the neck of femur. Additionally, patients presenting with spinal fractures only, tend to have both a higher number of spinal fractures than those presenting with a hip fracture and spinal fractures, although the severity of the individual fractures and the distribution of the fractures in the spine is similar. The bimodal trend of fracture location with an increased frequency around T7-T8 and another peak around T12-L1 is consistent with previous findings . This paMost studies evaluating the effectiveness of osteoporosis medications have focused on patients with poor trabecular bone, as reflected in low spinal density, with or without spinal fractures, and treated them with antiresorptives which are known to mostly act on trabecular bone. In general the effect on the prevention of further vertebral fractures has been impressive while the effect on non-vertebral fractures has been less so. Few studies have focused directly on the prevention of non-vertebral fractures in less selected patients and those that have, have not been very successful. Black and colleagues showed the presence of prevalent vertebral fractures indicated a high risk for future hip fracture whereas in the study conducted by McClung, the younger 70–79 year old group showed no benefit of treatment if the patients had no prevalent vertebral fractures ,18. LikeIt also appears that recruiting patients on the basis of a prior hip fracture without regard to trabecular bone status has limited benefit in the prevention of further hip fractures. For example, the zoledronic acid study of fracture prevention in hip fracture patients showed greater ability to prevent further  spine fractures and a more modest and non-significant benefit in the prevention of further hip fractures . UnfortuIf it is true that the presence or absence of trabecular bone mass reduction has an influence on the likelihood of response to treatment, it does not mean that trabecular bone loss is required for a hip fracture to occur, it simply increases the risk and may influence the type of fracture that occurs. Although the definition of osteoporosis has evolved to a risk paradigm, the notion of a t-score of −2.5 representing a significantly low BMD has to some extent survived, particularly at the spine site . Only a A prior “fragility fracture” is certainly associated with an increased risk of a further one, but this may be on the basis of a risk of recurrent falling. If there is no second fall, there will be no second fracture. Of note is the finding that the increased risk of a second fracture in those who have experienced a first fragility fracture, is largely independent of bone density, and even more so in older age, pointing to falling as the predominant risk factor . LikewisIt is notable that in our study the z-scores in the hip fracture patients without spinal fracture are normal  indicating no greater deficiency in skeletal mass in these patients than in the age and gender matched normative databases of the particular densitometers Table . Given tA potential weakness of the study is that the patients with hip fracture are a selected group of convenience. Patients missed, as noted above, are those who are younger and go directly home and those who go directly to LTC, who tend to be older. However as our comparison is with elderly ambulatory osteoporotic patients attending the osteoporosis clinic, the exclusion of a younger group, and an older, particularly frail group, is not inappropriate. Our study includes both genders on the assumption that hip fractures in either sex will show similar deficiencies regardless of gender. Although there are more women in the group with hip fracture and no spinal fracture, the analysis shows this to be group with the best bones, a finding which would be biased against by a predominance of women. On the other hand the spinal fracture alone group were slightly younger, but they turned out to have the most deficient bones. The analysis was re-run on the women alone. The smaller numbers made some comparisons fall below the significance level but overall the trends were unchanged.It appears that the risk of a hip fracture is a complex interaction of falling, person height, hip architecture and cortical and trabecular bone status, varying combinations of which determine the risk overall and, perhaps, the particular type of hip fracture sustained. It may be that only one of these factors, trabecular bone status, predicts a response to the current antiresorptives which are predominantly active on trabecular bone and the presence of vertebral fractures may be a simple indicator of such a state. More detailed reporting of the randomized controlled studies, in particular regarding the trabecular and spinal fracture status and the nature of the hip fractures that occur and the different responses to treatment in these various sub-groups, might bring better understanding of this phenomenon.The authors declare that they have no competing interests.RC conceived of the study and directed the data collection and wrote the manuscript. LC completed the data collection, performed the analysis and reviewed the manuscript. Both authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/14/68/prepub


The use of microwave imaging is becoming more prevalent for detection of interior hidden defects in manufactured and packaged materials. In applications for detection of hidden moisture, microwave tomography can be used to image the material and then perform an inverse calculation to derive an estimate of the variability of the hidden material, such internal moisture, thereby alerting personnel to damaging levels of the hidden moisture before material degradation occurs. One impediment to this type of imaging occurs with nearby objects create strong reflections that create destructive and constructive interference, at the receiver, as the material is conveyed past the imaging antenna array. In an effort to remove the influence of the reflectors, such as metal bale ties, research was conducted to develop an algorithm for removal of the influence of the local proximity reflectors from the microwave images. This research effort produced a technique, based upon the use of ultra-wideband signals, for the removal of spurious reflections created by local proximity reflectors. This improvement enables accurate microwave measurements of moisture in such products as cotton bales, as well as other physical properties such as density or material composition. The proposed algorithm was shown to reduce errors by a 4:1 ratio and is an enabling technology for imaging applications in the presence of metal bale ties. Applications range from detection of hidden weapons at airports to detection of dangerously high moisture areas in packaged and baled fiber products such as raw cotton lint. In applications for detection of hidden moisture, microwave tomography can be used to image a bale and then perform an inverse calculation –3 to derScattering of electromagnetic radiation from a sphere or long cylinder in which the sphere or infinitely long cylinder are significantly smaller than the wavelength, results in a scattering, quantified by classification as Rayleigh scattering, or as noted in radar handbooks, long thin wires that exhibit Mie scattering even though the diameter of the wire would normally place the scattering in the Rayleigh region –8. Thus,2.To quantify the influence of the metallic ties, used in packaging of the bales, The testing consisted of placing the bale at a static position in front of the conveyor and taking a frequency sweep to obtain scattering parameters in the S21 direction, as provided by an HP 8753D network analyzer utilizing an in-place air-path through calibration. The air-path calibration was obtained without the bale in the field of view and was utilized as a reference to remove the influence of the cabling, antennas and internal instrument variation and path lengths. After the air-reference was taken, the bale was then transported into position and a frequency scan was obtained for each position. After each scan the bale was indexed forward by 1 cm and the process was repeated. Thus, the testing obtained a frequency scan for each position as the bale was indexed past the antennas. The choice of the reported frequencies were chosen from the available frequencies that were determined to have an optimal standing wave ratio, “SWR”, match  with theThe second phase of the experiment repeated the bale scan with the replacement of the network analyzer with an ultra-wideband system configured with a 60 ps rise-time impulse generator and 12 GHz bandwidth sampling signal-correlator, such as is typical in ultra wide-band systems [For additional supporting evidence for this research, a finite-difference-time-domain “FDTD” electromagnetic simulation model, that was based on Yee’s seminal paper  and exte3.In examination of i.e., the combination of the scattered wave to the direct path wave is a function of wavelength and proximity spacing. Thus, this model supports the experimental results which shows fluctuations in the peak relative location in the measured phase delay, even thought the bale is of nominally uniform moisture and density across the entire volume of the bale.In working towards an understanding of the influence of the metal bale ties on the system, utilizing a first-order ray-tracing analysis of a multi-path signal propagation model , provideOf particular note is in the comparison of the Mie scattering magnitude response of To gain further insight into how the metallic bale-ties are influencing the signal propagation and hence the measured electrical permittivity, an FDTD simulation was conducted to compare the propagation for the case of the bale without metallic ties, versus without wire-ties, the cross-sectional area, depicted in red “cross-section” in To gain further insight into the impact of the two test FDTD cases, with The second phase of the experiment was a repeat of the bale scan experiment utilizing ultra-wideband, “UWB”, signals and time-domain processing. The original justification for the addition of the UWB testing to the protocol was to verify that the assumed phase delay integer-n roll-over, utilized for resolving the phase ambiguity for the network analyzer measurements, was correct. The testing however yielded the surprising results of near complete suppression of the metal bale ties from the received signal. A repeat of the experiment utilizing the same locations and correlated to the network analyzer scans provides strong support that the use of UWB signals can be used to remove the impact of the Mie scattering caused by the wire-ties. Qualitatively, in 4.The use of microwave imaging is becoming more prevalent for detection of interior hidden defects in manufactured and packaged materials. In applications for detection of hidden moisture, microwave tomography can be used to image a bale and then perform an inverse calculation to derive an estimate of the variability of the hidden interior moisture, thereby alerting personnel to damaging levels of unseen moisture before fiber degradation occurs. One impediment to this type of imaging is when the packaging utilizes metal strapping ties, as the reduced aperture of the imaging antennas, size and close proximity of the metal bale ties combine to create significant Mie scattering that causes destructive and constructive interference as the material is conveyed past the imaging antenna array.An experimental conveyance system was utilized that allowed for precise positioning of a bale with respect the sensing imaging antennas. By utilizing sequential measurements, where the bale is indexed forward to the next position, an incremental scan of the bale was obtained with measurements at multiple frequencies ranging from 500 MHz to 2.5 GHz. The experimental results revealed the expected variation due to the hypothesized multipath interference. However the experiment also revealed an unexpected deviation in the lateral variation in the peaks of the measurements that correlated to the wire-tie spacing. The experiments also revealed that each frequency would have to have a unique lateral offset from the metal bale-ties, which reinforces the earlier premise that a fixed channel filter would prove difficult to implement for the proposed removal of the multipath interference.This research examined the cause and effect from both theoretical aspects through the use of FDTD simulation models as well as experiments to support the theoretical conclusions. The research provides strong support to the hypothesis that the experimental variation in permittivity as measured on bales with uniform moisture profiles is due to Mie scattering off the metal bale ties. The research also developed a solution for the removal of the influence of the metal bale ties from the measurement through the utilization of UWB scanning in the time-domain or via stepped frequency domain scanning. It will also likely be possible for a frequency-modulated-continuous-wave, “FMCW” system to perform similarly when configured for ultra-wideband scanning of permittivity, rather than a narrow-band implementation. The development presented in this paper provides a method to significantly improve the accuracy by which measurements of moisture can be obtained for the use in the generation of microwave moisture images of bales tied with metal bale ties. This work is particularly relevant for low permittivity materials that have a very localized range such as is typical of cotton where the full range of expected relative permittivity ranges is typically limited to the range {1.4 to 2.5} as the spurious Mie reflections have a larger influence on the measured response.


The algorithm is intrinsically robust: with loss of communication among the vehicles the coverage performance  is degraded but not lost. The ensuing form of graceful degradation provides also a reactive measure against Denial of Service. The cooperative algorithm relies on the fact that the available information from the other sensors, though not necessarily complete, is trustworthy. To ensure trustworthiness, a security suite has been designed, specifically oriented to the underwater scenario, and in particular with the goal of reducing the communication overhead introduced by security in terms of number and size of messages. The paper gives implementation details on the integration between the security suite and the cooperative algorithm and provides statistics on the performance of the system as collected during the UAN project sea trial held in Trondheim, Norway, in May 2011.Methodologies and algorithms are presented for the secure cooperation of a team of autonomous mobile underwater sensors, connected through an acoustic communication network, within surveillance and patrolling applications. In particular, the work proposes a cooperative algorithm in which the mobile underwater sensors  respond to simple local rules based on the available information to perform the mission and maintain the communication link with the network ( The colOne scenario in which the use of multiple vehicles or sensor nodes presents critical aspects and peculiar characteristics is the underwater one. Many relevant infrastructures are placed very close to the sea or directly in the water, opening new scenarios for the use of Autonomous Underwater Vehicles (AUVs) and the development of Autonomous Ocean Sampling Networks (AOSN), where multiple nodes can cooperate as a group to achieve some common goals .Any cooperative mission necessarily involves communication among multiple agents. When the cooperating sensors are used in the underwater domain, communication issues become of paramount importance and in fact the agent spatial locations and mutual separation has a direct influence on the capability to communicate ,6. In thIn operative scenarios, not only is it necessary to share information to achieve the mission objectives, the ability to securely communicate also becomes a key issue so that the correct data is transmitted and received by the right agents, and only among the desired group. The possibility to share in a secure way the necessary information may in fact determine the success or the failure of the mission as a whole. Listening to private messages, or modification or injection of fake data are all usual threats in communication networks. They become even more critical in the context of distributed agents since cooperation may be achieved only when all the components receive the expected data from the legitimate peers. The underwater environment poses unique challenges also as far as network security is concerned, and again the traditional security mechanism successfully used and implemented on radio-based network suddenly becomes infeasible.emergent behaviour defining the action and the behaviour of each agent through simple elementary rules ).The cooperation among vehicles is modelled according to the group communication paradigm . VehicleWith reference to 3.This section focuses on the development of an adaptive cooperative algorithm for mobile sensor nodes, the moving components of the underwater network. When fixed sensors are present they will participate to the global performance, in terms of detection and communication, but since their position is fixed they cannot reconfigure and adapt themselves to varying scenarios. In this case, it will be the mobile agents’ responsibility to consider the presence of the fixed nodes, so as to guarantee that the final goal can be reached. Moreover, in order to allow a simpler description of the proposed method, in the remainder of this section, we assume the existence of an appropriate communication infrastructure which is reliable and robust enough to effectively allow a team of mobile agents to securely communicate and collaborate to achieve the required common goal. Further details on these aspects are given in Section 4.behavioral approach paradigm, into simpler subtasks (behaviors or rules) solvable in parallel hA) impliposed in , they ar4.The cooperative algorithms of the previous section critically relay on the trustworthiness of the transmitted information. This is achieved by means of two services, the Secure Dispatching Service (SDS) and the Key Management Service (KMS). The SDS is responsible for protecting confidentiality and authenticity of messages by encrypting and decrypting them as well as generating and verifying proofs of their authenticity. The KMS is responsible for revoking the current key and distributing a new one either periodically or upon a vehicle leaving.Implementing these services in a underwater acoustic network is challenging due to the severe limitations of the networking environment in terms of very high message propagation delay, very low bandwidth, and high energy consumption for communication. Limitation in the message size is hence of paramount importance in order to reduce battery consumption in autonomous nodes.4.1.eE(m||h(m)), where E specifies a symmetric cipher, h specifies an hash function and e is an encryption key. Alternatively, a transformation aimed at guaranteeing sole authentication of message m is m||aH(m), where a is an authentication key and H is a Message Authentication Code (MAC). The cryptographic suite, namely the actual cryptographic primitives that are used in a transformation, must be properly chosen because of the communication overhead it may imply.The SDS implements the cryptographic transformations that have to be applied to network traffic segments. A cryptographic transformation specifies the cryptographic processing to be applied to messages before sending or after receiving them. For instance, in order to guarantee both confidentiality and integrity of a message, a possible transformation is 4.1.1.Block ciphers split cleartext in blocks of fixed, predefined bit-length. In the most general case, cleartext length is not multiple of the block length. Thus padding is necessary. However, padding has the negative effect that the ciphertext may result up to one block longer than the corresponding cleartext. This effect is called ciphertext expansion. While ciphertext expansion is negligible in a traditional network, it becomes relevant in wireless sensor networks and, in particular, underwater acoustic networks. In these networks, communication and energy limitations require keeping a message size small and ciphertext expansion may introduce an overhead that is not negligible anymore. Let us consider the cooperation algorithm. At each step a vehicle sends 104-bit payload specifying the vehicle identifier (8 bits), position , and sonar detection range RD . Encrypting the payload by means of AES, whose block size is 128 bits, would introduce 24 bits of padding thus causing a 23% of payload expansion. Of course, for any given payload, the percentage weight of the ciphertext expansion much depends on the block size and ultimately on the cipher. However, in order to completely avoid the ciphertext expansion problem, we have used the CipherText Stealing (CTS) technique that alters the processing of the last two blocks of plaintext, resulting in a reordered transmission of the last two blocks of ciphertext and no ciphertext expansion .4.1.2.eE(m||h(m)) when it addresses both encryption and authentication [32 chances to blindly forge a digest. If an adversary repeatedly tries to forge it, he/she needs 231 trials on average. However, the adversary cannot perform trials off-line. This means that the adversary has to validate a given forgery only by sending it to an authorized receiver. This implies that the adversary has to send 231 messages in order to successfully forge a single malicious message. In a conventional network this number of trials is not large enough. However, in a underwater acoustic network this may provide an adequate level of security. An adversary can try to flood the network with forgeries, but on a 2 kbps channel with 184-bit messages, he/she can only send about 11 attempts for second. Thus, sending 231 messages requires around 75 months, i.e., about 6 years. Battery-operated vehicles have not enough energy to receive that many messages. Furthermore, the integrity attack would translate into a denial of service attack since the adversary needs to occupy the acoustic channel for a long time. Fortunately, it is feasible to detect when such an attack is underway. UAN uses a simple heuristic: vehicles could signal the base station when the rate of digest/MAC failures exceeds some predetermined threshold.Encryption without authentication is insecure . For exatication . More sptication ,23. Secutication . An adve4.2.Ki)( by verifying that Ki)( = h(Ki+1). To reduce the communication overhead, KMS maintains a logical key tree  rekeying messages, where n is the number of vehicles. Furthermore, given the key-chain authentication mechanism, every rekeying message needs to carry only the next key (in its encrypted format). No additional information proving key authenticity is thus required. Notice that this is a great advantage in terms of communication overhead with respect to using digital signatures, for example. Let us assume that group keys are 128-bit long and we use ECC-180 digital signature to authenticate them. ECC-180 is nowadays considered as secure as RSA-1024. In ECC-180 a digital signature is 360 bits and thus a rekeying message would be 488 bits, i.e., 3.8125 times longer than in the approach proposed here.Upon receiving a rekeying message, after it has been properly decrypted, the authenticity of the next key therein contained is verified by computing its hash and comparing the result to the corresponding current key. For instance, upon receiving rekeying message 5, 5.This section report results from the application of the above mentioned algorithms to the UAN project experimental activities held in Trondheim, Norway, in May 2011. The network see  was compThe message expansion caused by the authenticator which in turn increases the probability of packet loss.A decrease in the acoustic communication conditions.The presence of such a mobile agent allowed for change in the network geometry to tackle changes in the environment. During the test, which involved several different communication objectives not covered in this work as they would go beyond the scope of the paper, the vehicles were used as data-relays to improve and to re-establish broken communication links between fixed nodes  and they were integrated into the UAN wide-area protection system for the protection of a high value asset co-located with the UAN base-station. Even though it is not possible to evaluate the specific weight of each of the two components in the mix, the ADR decrease is sustainable and the effect of the use of network security appears not to be critical with respect to the decrease in performance due to the degradation of the communication channel.6.i.e., the mission goal) is degraded but not lost. Moreover, the algorithm attempts to minimize the information exchange among the vehicles prior to the local decision. Area coverage performance of the proposed adaptive approach has been discussed and compared with those obtainable with optimal geometrical solutions. It was also shown how, in dependence with the type of information transmitted but without introducing additional data exchange or algorithmic complexity, the proposed algorithm may instigate additional and more complex team behaviors. Crucial for the agents cooperation is the trustworthiness of the messages among the sensors. To ensure it, a security suite based on the group communication paradigm has been designed, at the communication middleware level. The security suite has been specifically oriented to the underwater scenario, and in particular to the goal of minimizing message size overload and computational and message exchange increase, while still guaranteeing security. Details on the implementation of the given methodologies have been given, describing the architecture developed to integrate the cooperative algorithm into the security suite. Furthermore, we reported statistics and figures of merit on the performance of the UAN project underwater acoustic network as tested in May 2011, in the Trondheim area, Norway. The UAN experimental testing was focused on the general validation of the network architecture and it included all the security mechanisms described in this work and the autonomous adaptation and cooperation of the nodes.The paper has described a methodology for secure cooperation within a network of autonomous mobile underwater sensors connected through an acoustic communication network. A cooperative algorithm based on the behavioural paradigm has been illustrated. Each mobile sensor solves simple parallel subtasks responding to local rules based on the available information to perform the mission and maintain the communication links within the network. The algorithm has been designed to be intrinsically robust, in the sense that with loss of communication among the vehicles the coverage performance (


Wolbachia and Cardinium, have been well studied through field data and validations on the basis of numerical simulations. However, the analytically derived equilibrium frequency of multiple infections has not yet been determined, although the equilibrium for cases of single infection has been reported. In this study, we considered the difference equation for endosymbionts using three parameters: the probability of the failure of vertical transmission (Cytoplasmic incompatibility (CI)-inducing endosymbiotic bacteria, such as  Wolbachia and Cardinium are well known to be reproductive manipulators infceting insect cells Drosophila simulans individuals were infected with Wolbachia in the middle of the 1980s in California; however, the infection rate increased to 95% in 1993 Encarsia pergandiella infected with Cardinium, the infection gradually spread to almost fixation within the population, irrespective of the initial rate of infection Endosymbiotic bacteria such as The dynamics of CI-inducing bacteria can be primarily determined by three parameters: vertical transmission efficiency, fecundity of infected females, and CI levels Wolbachia, using the three conventional parameters Wolbachia in parasitic ants (nine strains) Models have been developed to study the dynamics of CI-inducing bacterial symbionts, particularly in Wolbachia infections In addition, the modeling of endosymbiotic dynamics in the context of mating system has not been extensively studied. CI is a phenomenon that occurs via mating, thus it is considered that the mating system should affect the dynamics of the endosymbionts. Mating systems can be categorized roughly as random mating and inbreeding. Random mating or panmictic mating is mating between any individuals in the population. In contrast, inbreeding or sib mating, is mating between a brother and a sister. Therefore, CI should be less effective under inbreeding because the mating partner should have the same infection status. Strong inbreeding has been observed in some insects, such as fig wasps and ambrosia beetles, which are known to have This study aimed to develop an analytically solvable model of the infection dynamics of CI-inducing bacteria in sib/panmictic mating systems. We derived the analytical equilibrium of single and double infections, and generalized arbitrary numbers of strain infections using three parameters . Our results show that bacterial infection thresholds can be simply expressed as a function of the ratio between the failure of vertical transmission and the CI level. In addition, we derived the maximum number of bacterial strains that are capable of infecting a host population.We consider a host population infected with We develop an difference equation to express the dynamics of the frequencies of hosts infected with multiple bacterial strains. Let Next, we consider the formulation of CI among strains. CI results in a decreased hatching rate by a factor A bacterial strain can be transferred by vertical transmission from a mother to offspring at egg stage with a probability of The occurrence frequency of CI depends on the mating pattern. Under random mating, CI occurs depending on the distribution of Frank also proved that a polymorphic equilibrium, where both infected and uninfected individuals exist, can be stably maintained only if there are host individuals infected with all of the bacterial strains in the population possible . Frank sNext, we analytically derive the equilibrium for the system . We willWhen e system  can be rBecause unstable . The unsIn the case of e system  can be rAs stated in the previous section, we assume that d  than  .Finally, we calculated the fixed points of sumption . For exaured at  . The redWe compared the analytically derived The inverse function of In this study, we demonstrated the analytic equilibrium solutions of the frequencies of individuals infected with all the bacterial strains in a population with single, double and arbitrary Our model used a parameter The analytically derived equilibrium depends mainly on one parameter, is lower . In thisWolbachia-induced speciation. Suppose that Our theoretical results suggest that the equilibrium frequency of completely infected hosts decreases as ncreases . In addincreases . The sevWolbachia and Cardinium obtained using natural populations or an artificial line  that infect Callosobruchus chinensis are variable Drosophila simulans infected with four Wolbachia strains in Madagascar. The CI levels of the hosts vary between 0 and 1, depending on specific crosses In this study, we aimed to provide a qualitative outline of the dynamics of multiple infections by introducing an analytically solvable model. By ignoring quantitative accuracy, our model sheds light on the mathematical structure related to the multiple infection dynamics. However, we introduced several assumptions to develop a solvable model, which should be eliminated to allow more quantitative comparisons using experimental data. First, we assumed that a common transmission rate or CI level were shared among bacterial strains. If the bacterial parameters are heterogeneous, a bacterial strain with a lower Figure S1Dominancy of highly-infected individuals. (A) An example of distribution of frequencies (EPS)Click here for additional data file.Table S1The values of parameters and the frequency of completely infected hosts in the field. These values were reported in the references listed above.(EPS)Click here for additional data file.


This major result suggests that the adhesion of the particles to the substrate is strongly reduced by the presence of hydrophobic interfaces. The influence of critical parameters on the manipulation was investigated and discussed viz. the shape, size and grafting of the NPs, as well as the surface chemistry and the patterning of the substrate, and finally the operating conditions . Whereas the operating conditions and substrate structure are shown to have a strong effect on the mobility of the particles, we did not find any differences when manipulating ordered vs random distributed particles.One key component in the assembly of nanoparticles is their precise positioning to enable the creation of new complex nano-objects. Controlling the nanoscale interactions is crucial for the prediction and understanding of the behaviour of nanoparticles (NPs) during their assembly. In the present work, we have manipulated bare and functionalized gold nanoparticles on flat and patterned silicon and silicon coated substrates with dynamic atomic force microscopy (AFM). Under ambient conditions, the particles adhere to silicon until a critical drive amplitude is reached by oscillations of the probing tip. Beyond that threshold, the particles start to follow different directions, depending on their geometry, size and adhesion to the substrate. Higher and respectively, lower mobility was observed when the gold particles were coated with methyl (–CH By the M in UHV . In addiM in UHV . UnfortuM in UHV –10. Chan60 islands on NaCl was performed by Lüthi et al. using contact AFM [2 surface [pushed on a graphite surface by the AFM tips and then manipulated by placing the AFM tip on top of the particles. Above a certain lateral force threshold, particle sliding was observed, which has allowed the transition from static to kinetic friction to be quantified [The first mode used in AFM was the contact mode. Manipulation of large Ctact AFM . Even iftact AFM . Byungsotact AFM  have alstact AFM –15. For  surface . Nanomet surface . These pantified .A compromise between the contact and non-contact AFM techniques is the intermittent mode, the so called tapping mode. In this mode the phase shift of the cantilever oscillations with respect to the external periodic excitation can be used to estimate the dissipated energy during manipulation. This method was recently used by Ritter and coworkers to manipulate antimony particles on a graphite surface in air –18. PaolFor this reason, colloidal particles have appeared as model nano-objects because they can be produced in various well-controlled sizes and from various materials such as metals or semiconductors. Of particular interest has been the use of metal nanoparticles, which have been applied to the development of highly sensitive nanoparticle-based detection assays that utilize electrical or optical detection (colorimetric and surface enhanced Raman spectroscopy (SERS)). For different reasons gold particles are particularly attractive in this field. For instance, they are ideal electrodes for molecular electronics . Gold clT and relative humidity RH%, are presented and discussed. The dependency of the energy dissipation during the manipulation was particularly studied as a function of size, coating of particles, substrate and temperature. Finally, interpretation of the physico-chemical mechanisms involved at both interfaces – tip–particle and particle–surface – during the movement of the particle was proposed and partially verified by modeling; nevertheless additional investigations are still needed.In this paper, the sensitivity of those critical parameters on the mobility of gold nanoparticles during their manipulation using AFM in tapping mode has been investigated. In particular, the effects of the size, shape and coating of the nanoparticles, the lateral scan velocity, the particle-surface interactions and the environmental conditions, especially temperature Spherical and asymmetrical gold nanoparticles were synthesized as described in the Experimental section and deposited onto flat and patterned surfaces. Accurate manipulation was performed using AFM in tapping mode as it provides indirect access to dissipation energy during particle movement . Since tT, RH%), surface topography and tip scan velocities on the manipulation performance of gold nanoparticles. Finally, conclusions with discussions and future directions are given in the last paragraph.The ﬁrst part of the discussion will focus on the influence of the size and shape of the particle on manipulation. Then, we will examine the effect of functional (hydrophilic vs hydrophobic) molecules grafted on the Au nanoparticles on their mobility. In addition, we will address the important issue of environmental conditions (1.−), referred to as “reference NPs” were deposited onto bare and hydrophobized (CH3-terminated coating) silicon wafers, and manipulated using AFM in tapping mode. During manipulation, the oscillation amplitude of the tip, Aset, was kept constant by a feedback loop. In such cases, the power dissipation accompanying the tip-sample interaction can be determined from the following relationship [Sizes of gold spherical nanoparticles(NPs) were tuned from 5 nm up to 65 nm according to the synthesis procedure described in the Experimental section. “As-synthesized” Au NPs, meaning NPs covered with citrate stabilizing group  was performed for 5 sizes of Au colloidal particles whose radius (R) was varied from 5 up to 65 nm. The calculation of the dissipated power .Indeed, it has been observed  that twoMoreover, the modeling of the NPs trajectory addresses a relation between the frictional forces acting on spherical nanoparticles, and the trajectories predicted. This model can also be used to interpret the trajectory fluctuations and the apparent discontinuities observed when spherical gold particles are manipulated on rigid substrates by AFM.The manipulation of spherical and asymmetrical nanoparticles by AFM represents a way to understand and control the motion of complex shaped nanoparticles. For instance, manipulation of elongated objects such as rigid Au nanorods induces mainly sliding and rolling of the nano-objects, and this movement varies with the different stages of nanomanipulation time scale. As shown in For triangular and flower shaped nanoparticles, the nano-objects mostly evolve through a translation movement, as well as a rotation along their main perpendicular axis during the manipulation, as shown in 2.Because real surfaces are often heterogeneous in their chemical composition, functionalized nanoparticles provide good model systems to study and tune the mobility of nano-objects on these substrates. As a next step, the role of the hydrohilicity and hydrophobicity of the functional grafting on spherical Au nanoparticles is illustrated in 3-terminated thiol groups  and moving these particles against a flat bare silicon substrate. The results are summarized in 3-coated particles. We also observed that the manipulation of hydrophilic coated nanoparticles often results in a damage to the tip due to the high particle–substrate adhesion force. This strong adhesion between silicon substrate and hydrophilic coated nanoparticles primarily arises from intermolecular interactions. It may also involve a contribution from capillary bridges between the substrate and the NPs on one hand and between the closest NPs on the other hand . In contrast, it has already been observed that the thin adsorbed water film formed on the silicon wafer acts as a lubricant when confined between the hydrophobized CH3-coated nanoparticles and the (hydrophilic) substrate [The role of the hydrophobic or hydrophilic character of the interface in the manipulation process was investigated, using gold nanoparticles bearing OH- and CHubstrate –43.RH%) which is an environmental parameter, strongly depends on the chemistry of the NP–substrate interface. Another environmental parameter, namely temperature, also affects the mobility of the nanoparticles. The influence of extrinsic  parameters is discussed in the following paragraph.As we can see here, the eventual role of relative humidity (3.kBT) impedes the formation of stable intermolecular bonds and water bridges between particles and substrate, reducing the adhesion between them. Similar thermal effects have been recognized in friction on hydrophilic surfaces measured with different scan velocities [locities . It is wEact/kB), where ΔEact represents an activation energy barrier with respect to a reference state E0: ΔEact = (E0−Eact) where Eact(T) is the energy input involved in the motion of the particle. This energy variation (slope) is high for the CH3-hydrophobized NPs, indicating a strong decrease of the input energy with the temperature which would be expected for low adhesion strength between nanoparticle and substrate. Surprisingly, a quite similar behavior in, both, trend and activation barrier of the temperature-dependent mobility is observed for the hydrophilic OH-coated NPs. An explanation for this result may come at least partly from the complex behavior of the adsorbed  water depending on temperature in the hydrophilic system. Beyond the observed – and rather reasonable – general trend, the strong decrease with the temperature of the energy required for particle movement, the magnitude of the activation barrier for essentially hydrophilic and hydrophobic contacts will certainly need further confirmation experiments, as well as a more extensive interpretation. Indeed, we assumed in our treatment −1/2 is larger than the range of short-ranged forces [The power dissipation at different temperatures is comparable in both cases. This result can be explained by the average distance between the nano-objects, which is 70 nm for the random distribution and 100 nm for the ordered one. At such a scale, the interparticular forces are of the order of long range interactions. The mobility of particles is essentially affected by electrostatic interactions arising from residues from the synthesis (citric acid) that may be adsorbed on the particles. It is thus normal, in the absence of both physical contact and notable intermolecular forces between the particles, that their mobility is independent of their organization (random or ordered). In other words, this result means that as long as the particle number density d forces , their m5.The presence of surface contaminants (dust or water) affects the mobility of nanoparticles as this directly changes the intermolecular interactions between the nanoparticles and the surface. As it has been discussed in subsection 2, a contribution from capillary bridges has also a strong influence on the mobility of spherical Au nanoparticles during their manipulation. Indeed, capillary forces of water films between both interfaces, nanoparticle–surface and tip–nanoparticle, will depend on the volume of liquid condensate present at the interface, as well as the interface geometry  see Sch. The pre3-coated ones. The diameter of the Au nanoparticle is about 20 nm. The ambient (RH = 33%) and higher relative humidity results displayed in In this section we describe our studies on the mobility of as-synthesized Au spherical NPs and CHRH = 43%, the adsorbed Au particles do not move, because the energy transferred from the tip to the particle during the tap is not high enough to break the capillary bridges formed at both interfaces. As a consequence, the overall energy does not reach the threshold barrier to move the particle and is completely dissipated in the system.The decrease of relative humidity from 53 down to 33% has a strong impact on the mobility of the hydrophilic Au NPs. Above However, this process does not affect strongly the mobility of hydrophobic Au NPs. They move whatever the environment. This difference can be explained by the existence and the local shape of a liquid condensate  around tIn a humid environment, the magnitude of friction and adhesion forces is strongly dependent on the capillary force that is related to the intrinsic wetting properties of the interfacial system. As a consequence, the resulting water meniscus (or layer) can either increase friction through increased adhesion in the contact zone (hydrophilic interfaces) or reduce it through the lubricating effect of a water layer.Further experiments should also prove that the bigger the particles are, the higher the capillary effect will be as has previously been observed in contact mode –48.The environment is a crucial parameter in manipulation  experiments. The adhesive and frictional results are directly dependent on the humidity and temperature of the surrounding medium. Concerning the influence of humidity (or more exactly the absence of humidity), we have investigated how the nanomanipulation process is affected in ultra high vacuum (UHV) environment. The topography image in The previous sections have demonstrated the influence of the morphological, environmental and chemical parameters on the mobility and movement of the particle. The following addresses the influence of the topography of the substrate.6.Manipulation of gold nanoparticles was investigated on flat bare silicon wafers, as well as on nanostructured (or nanopatterned) silicon wafers, i.e., silicon substrates that are patterned on the nanoscale.The following experiences were performed using a raster scan path of the tip mounted on a Nanosurf AFM. On flat bare silicon wafer, the direction of motion of the 25 nm diameter gold nanoparticles was initially well defined, but changed after acquiring a couple of images. This makes it much more difficult to move the particles, even for higher values of the drive amplitude, possibly because of tip contamination. Hence, the idea to modify the topography of the surface was chosen to study the effect of the geometrical surface confinement on the mobility and trajectory of the nanoparticles. Nanopatterned substrates shown in b separating two scan paths on the deflection angle has been shown by simulation of these experiments [The surface patterns consist of an array of nanopits created by the focused ion beam (FIB) milling technique. The width and depth of the pits are 650 nm and 5 nm, respectively, and the spacing between two adjacent pits is 125 nm. On the patterned surface, the mean direction of motion remains identical (on average), even after a long acquisition time. This stability of the direction of the particle movement observed here on the nanopatterned substrates can be attributed to "self-cleaning" of the tip when it crosses the shallow pits. Considering that the pits have only a small influence on the particle direction , which meriments –49. Figueriments .Manipulation experiments were repeated to check the influence of the deep grooves (either on Si wafers or on HOPG) on the trajectory of the moving particles. It was found that the deep grooves slightly influence the direction of movement of the particles as particles tend to follow their preferential angle during movement.Finally, the last important and technological parameter of AFM nanomanipulation is the effect of scan velocity on the movement of the nanoparticles.7.The inﬂuence of the sliding velocity on friction, which accounts, at least partly, for the dynamical response of the boundary layer, can be exploited to gain insight into the manipulation of nano-objects .−1 on three model substrates, i.e., a cleaned silicon wafer (SiO2), and two other ones, coated with either hydrophilic (–NH2) or hydrophobic (–CH3) self-assembled monolayers.Spherical particles (as-synthesized Au NPs) of 35 nm and 60 nm in diameter were moved in tapping mode with Veeco AFM following the previous procedure described in subsection 1. The drive amplitude threshold to move the particle was recorded as well as the phase shift to estimate the loss of energy during the movement of the particles. These experiments were repeated for different scan tip velocities ranging from 0.1 up to 10 µm·sThe results of the velocity-dependence of the dissipated power are plotted in Our results in 3), the interactions with the hydrophilic nanoparticles (as-synthesized citrate stabilized NPs) mainly involve London dispersion forces that have a much lower magnitude as compared to the polar, hydrogen and electrostatic bonds involved in the adhesion of these citrate-stabilized nanoparticles, with more hydrophilic (SiO2 and NH2) substrates. The maximum dissipated power appears for the more polar substrates. It is worth noting that this value can involve a contribution from the capillary water bridges which readily form on more hydrophilic systems under ambient conditions as previously discussed in subsection 5A. It is also worth noting that we also verified here that both the surface and the particle were free of any observable damage after each manipulation.At the more hydrophobic substrate -dependent viscoelastic and/or plastic dissipation in polymers , as is described for instance through the time-temperature superposition principle for polymers –52. HoweRH% and temperature T), as well as the tip scan velocity. The dissipated power during manipulation was quantified under various operating conditions . Our experiments show that the velocity dependence of the dissipated power at these nanoscale contacts is far more complex than what one could predict, based on the sole contribution of the tap energy and capillary liquid bridging adhesive force. Indeed, the thermal energy produced within the tip–substrate contact can induce molecular excitations and structural transitions in the topmost contacting layers, the magnitude of which also increases with the sliding velocity. Direct access to the nanoscale contact between tip and nanoparticle, and nanoparticle and surface are limited with the current device, thus any quantitative analysis of these results remain at this stage scientifically debatable. The second difficulty is naturally related to the yet insufficiently understood size effects that show up in nanoscale friction and strongly affect the results. In addition, real-time monitoring of the manipulation process is almost impossible. Most of the time, imaging is offline and the unexpected problems during pushing cannot be detected. Another way is utilizing the force feedback information during pushing for reliable manipulation. This is currently being seriously investigated and correlated to theoretical studies [The manipulation of nano-objects is still a relatively rare operation. Because micro/nanomechanics has not been completely well-developed, two-dimensional positioning of nanometer-size particles on a substrate at ambient conditions remains a difficult operation and depends on several critical physical, mechanical and chemical parameters. However, advances have enabled better control in nanoscale manipulation. In this paper, we have described manipulation of gold colloidal nanoparticles using AFM in tapping mode. The inﬂuence of structural characteristics of the particle  and the substrate (chemistry and topography) have been investigated. It has been shown that the mobility of the particles was significantly affected by the nature of intermolecular tip–particle and particle–surface interactions, the particle shape and size, the operating environment conditions (relative humidity  studies . Because3) or hydrophilic groups .Gold nanoparticles were adsorbed onto silicon wafers and manipulated in AFM tapping mode. They were either bare or coated with self-assembled monolayers terminated with hydrophobic , which, by reducing HAuCl4, imparts the negative charge of the citrate ions to the gold nano-particle surface [The colloidal suspension was made by reduction of an aqueous solution of nanogold particles, HAuCl surface –28. The Dodecanethiol for methyl terminated monolayers and 11-mercapto-1-undecanol for hydroxyl terminated monolayers were obtained from Sigma-Aldrich and used as received. Hydroxyl or methyl-thiol-stabilized gold nanoparticles were synthesized according to a modified version of two common syntheses . The as-For the adsorption experiments, a concentration of 0.03 wt % of nanoparticles in the aqueous or organic dispersion was used. The experimental protocol basically involved the particle adsorption by immersing the samples for about 20 minutes in the suspension, whose temperature was maintained at 20 ± 1 °C. After this initial adsorption stage, the samples were removed from the bath, and the thick dispersion film remaining at the substrates was allowed to dry.block-poly(2-vinylpyridine). The solution was deposited onto silicon wafer and dried under a nitrogen flow. After being dip-coated, the polymer was removed by oxygen plasma treatment (see Samples were provided by McFarland’s group at UCSB. Au nanoparticles (25 nm diameter) were synthesized as described previously –28. The ment see .2) layer of ~1.5 nm. The organosilane compounds were methyl terminated hexadecyltrichlorosilane (–CH3), and the amine terminated 6-aminohexylaminopropyltrimethoxysilane (–NH2). Homogeneous films were obtained by vapor-phase deposition in a dynamically evacuated chamber (1 h at 10−3 torr), using a mineral oil as dispersing solvent for the molecules. This consists of mixing the organosilanes in paraffin oil before evacuating the atmosphere in the dessicator enabling the molecules to pass into the vapor phase and stick to the substrate placed above the mixture [The molecular surfaces were prepared by self-assembling organosilane molecules onto silicon wafers Si(111) with a native thin oxide  were used. During manipulation, the oscillation amplitude of the tip, Aset, was kept constant by a feedback loop. In this case, the power dissipation accompanying the tip–sample interaction can be determined from The images in air were acquired with two commercial AFMs . Rectangular silicon cantilevers with resonance frequencies −9 mbar. Due to the high quality factor in UHV, the out-of-contact-resonance frequency shift was used as the imaging parameter instead of the tip's oscillation amplitude (NC-AFM). We have also performed measurements in contact mode, where the set point is determined by the normal load acting between tip and sample. PPP-NCLR and CONT cantilevers from Nanosensors were used in both cases.The images in UHV were acquired with a custom built AFM available at the University of Basel . The bas


Recent behavioral neuroscience research revealed that elementary reactive behavior can be improved in the case of cross-modal sensory interactions thanks to underlying multisensory integration mechanisms. Can this benefit be generalized to an ongoing coordination of movements under severe physical constraints? We choose a juggling task to examine this question. A central issue well-known in juggling lies in establishing and maintaining a specific temporal coordination among balls, hands, eyes and posture. Here, we tested whether providing additional timing information about the balls and hands motions by using external sound and tactile periodic stimulations, the later presented at the wrists, improved the behavior of jugglers. One specific combination of auditory and tactile metronome led to a decrease of the spatiotemporal variability of the juggler's performance: a simple sound associated to left and right tactile cues presented antiphase to each other, which corresponded to the temporal pattern of hands movement in the juggling task. A contrario, no improvements were obtained in the case of other auditory and tactile combinations. We even found a degraded performance when tactile events were presented alone. The nervous system thus appears able to integrate in efficient way environmental information brought by different sensory modalities, but only if the information specified matches specific features of the coordination pattern. We discuss the possible implications of these results for the understanding of the neuronal integration process implied in audio-tactile interaction in the context of complex voluntary movement, and considering the well-known gating effect of movement on vibrotactile perception. In everyday natural situations the combination or integration of multiple senses is essential for an adapted goal directed behavior It is well known that detection of tactile events is reduced during the execution of movements, a gating effect We selected the so-called three balls cascade juggling trick, for which the tactile suppression phenomena has recently being demonstrated What type of external periodic stimulations could improve the performance at juggling?Sensory stimulation can increase the robustness of elementary coordination against internal biological noise or external perturbation To provide the juggler with two metronomes without overloading vision, we assumed that non-visual cross-modal pairings matching the multi-frequency nature of juggling would be the best stimuli to improve the stability of the coordination. Accordingly, we focused our study on audio- tactile parings. Previously important principles underlying the combination of sensory cues from different modalities have been discovered, notably that integration takes place according to a statistical optimal rule In the present study, our prediction that one can stabilize the juggling coordination with adequate external sensory stimulations was tested using periodic audio-tactile stimuli, respectively presented at the ears and at the wrists, and providing specific tempo. Because balls and hands frequencies are core to the multiple component coordination in juggling, we chose to associate each sensory modality to one of these main components. The frequencies of the tactile metronome and of the auditory metronome were thus scaled to match, respectively, the tempo of the hands and the tempo of the balls. In one condition, the metronome frequencies were multiplied by two to examine the generalization of the parametric stabilization to an audio-tactile pairing Seven right-handed students  from the Montpellier 1 University participated in this study. Each participant signed an informed consent form approved by the Institutional Review Board (IRB) of the Montpellier 1 University (UFR STAPS).Because of the very low natural variability of expert jugglers rd metacarpal, to record the left and right hand movements. The three balls were covered with reflective tape and defined as markers, to record the movements of the balls.Data were collected using a Vicon 3D motion recording system at a 100-Hz sampling rate. The system was calibrated according to the manufacturer's instructions prior to data collection. Two reflective markers were taped on the dorsal head of the 3Thanks to additional markers fixed on each shoulder, we had able to follow the rotation and translation movement of the subject in the environment during the task. To homogenize the set of trials, we realized a projection of the data, first recorded in the lab frame, into a new reference frame relative to the subject . The stimulation was delivered with a DC motor (weight: 42 g) strapped on the ventral part of each wrist. Preliminary tests showed that the time to reach 100 Hz was less than 5 ms.The auditory metronome consisted in 80 ms square wave pulses (tone carrier frequency: 300 Hz) delivered to headphones fixed on top of the auricles. Headphones also played white noise in order to isolate the subject from the noise emitted by the vibrotactile stimulation.We wanted to compare the effect of metronomes that differed in modality , frequency (simple or double) and, in the case of simple frequency metronomes, in the phasing of the stimuli (in-phase or anti-phase).Running the full crossing of the three factors in a repeated measure design would necessitate multiple sessions per participant. As a consequence, we selected the most interesting metronome conditions. Because metronomes that beat twice per movement generally provide a better guidance More precisely, because the juggling is the assembly of two frequencies within a single task We used a repeated measures design, where each participant had to juggle in each one of the 6 metronome conditions. The order of the metronome conditions was randomized across participants. In each metronome condition, participants had to perform a bloc of 5 trials.The experimental session started with a preliminary trial to record the preferred juggling frequency of each participant. Then, during 10 minutes, the participant was familiarized with the 6 metronome conditions. After this familiarization, the participant performed a block of 5 trials in each one of the 6 metronome conditions, for a total of 30 experimental trials.No instructions to synchronize with the stimuli were given, and participants were instructed to start juggling after the metronome started.The task was to juggle as regularly as possible for the duration of a trial: 20 to 30 s .For each participant individually, the frequencies of the metronomes were set at her/his preferred hands frequency (or twice the preferred frequency in the Doub. conditions).The marker data were dual passed through a second-order Butterworth filter (fourth-order) with a low-pass cutoff frequency of 8 Hz prior to further data processing.From the vertical velocity profiles of balls, we identified the moments of throw and catch: a positive velocity peak denotes a throw, and a negative velocity peak denotes a catch.We then used the average period of time between two consecutive throws by the same hand to compute the hand frequency (Ωhand).We addressed the organization of the juggling by analyzing the variability of the juggling pattern among the metronome conditions. Variability was computed as the within-trial standard-deviation of the variables of interest, using directional statistics for angular values First, because the timing of balls events seems to be the focus of control in intermediate and expert jugglers Second, we addressed the organization of the juggling in space via the within-trial variability of the distance between two consecutive throws in the vertical plane  and, if the coupling is strong enough, in systematic phase relations  between the hands and the metronome Frequency locking was easy to assess from a frequency synchronization index (FSI) computed from the difference between the observed hand frequency and the frequency of the metronome see .Phase locking necessitated a more sophisticated approach. For each hand, the instantaneous phase was estimated using the Hilbert's transform of the vertical positions time series Furthermore, we calculated the dwell time of the relative phase , chap.4.Operationally, to minimize the effect of noise after differenciation of the relative phase, we smoothed the time series of slope with a 4-points moving-average before calculating the proportion of time spent in small-slope episodes . We tried four different limit values for a small-slope episode . The dwell time (DT) is the time spent in small-slope episodes divided by the total duration of the trial and multiplied by 100 to get a percentage.To assess the significance of the effects of the metronomes on the juggling performance, we analyzed the variability of the juggling and, in a second step, focused on the synchronization to the metronomes, in relation to the juggling performance.p values after Huynh-Feldt correction for non spherical variance.All analyses of variance were complemented with Tukey HSD tests for post-hoc mean comparisons, and we reported the raw F values and degrees of freedom, but In all statistical analyses, the significance level was set at .05.Participants clearly differed in their level of variability in the control condition, which corresponded to differences in their juggling experience, despite the inclusion test and criteria. Accordingly, to improve the sensibility and reliability of our analysis to distinguish the five metronomes conditions, we subtracted for each subject his/her mean variability measured in the control condition to the variability measured in the metronomes conditions, and this for each given variable. Thus in this part of the analysis all variables were baseline corrected.t-test. As we emphasized the sensitivity of the analysis, we did not use a Bonferroni-Sidak correction p value threshold set at .05.To examine the effect the 5 metronome conditions on the juggling performance, we ran a global analysis of variance with repeated measures (ANOVA). Moreover, in order to test for significance against the baseline level, we compared the juggling performance under each metronome condition against the hypothetical mean of zero, the latter corresponding to the baseline level of variability, using a one sample To globally address the issue of synchronization, we analyzed the timing of the juggler's hand relative to the metronome beats, using an ANOVA with repeated measures, as previously.In a last step, being aware that mean frequency differences between movements and the metronome presented (FSI) varied between subjects, we further examined the phase synchronisation to the metronomes in each participant individually. We ran a within-participant statistical analysis using a surrogate approach to distinguish true phase synchronization from values obtained by chance. One type of synchronization, a weak synchronization, can indeed be expressed by a correspondence between the mean frequencies while phase synchronization is absent Because larger values indicate better synchronization in the surrogate distributions obtained for each participant, we took the 95 percentiles as the threshold value to get a p value of 0.05 for significant synchronization Finally, we analyzed the correlation between the synchronization indexes and the variability of the juggling pattern, with the prediction that better synchronization to the metronome would render lower variability in the juggling pattern.The first step in the analysis of the data was to examine whether the temporal information carried out by the external metronome affected the throwing action. The ANOVA applied on the variability of the initial angle at throws (ThAngle) was inconclusive.4, 24 = 3.03, p = .037). It especially showed that the velocity variability with the antiphase audio-tactile metronome (Multi. Anti.) was smaller than the variability obtained with the unimodal tactile metronome (Tact. Doub.).The ANOVA on the variability of the velocity at throws (ThVel) indicated an effect of the metronome condition .In addition the t-test comparison showed a variability of the velocity at throws in the unimodal tactile condition (Tact. Doub.) significantly larger than zero . It evidenced a smaller variability of the BFT in the antiphase audio-tactile condition (Multi. Anti.) than in the unimodal tactile condition (Tact. Doub.).The ANOVA on the variability of the balls flight time (BFT) revealed a significant effect of the metronome condition . The antiphase audio-tactile condition (Multi. Anti.) decreased the DBTz variability when compared to the in-phase audio-tactile condition (Multi. Simp.). In addition, let us remark that post hoc comparisons suggested, with a probability very close to the threshold for significance (p = .051), that this variability was smaller with the audio-tactile double metronome (Multi. Doub.) than with the audio-tactile simple metronome (Multi. Simp.).The ANOVA on the variability of the vertical distance between two consecutives throws (DBTz) also showed a significant effect of the metronome condition .Finally, t-tests showed that the variability of DBTz reached in the antiphase audio-tactile condition (Multi. Anti.) was significantly smaller than the zero baseline level corresponding to the control condition (tThe ANOVA on the frequency synchronization index (FSI) was not conclusive.In addition, neither the ANOVA on the phase synchronization index (PSI) nor the ANOVA on the dwell times (DT) showed a significant effect of the metronome conditions.However, as the differences between the actual average hand frequency and the metronome frequency varied between participants, this may have overridden the detection of differences in phase synchronization between metronomes conditions. The individual statistical analysis based on surrogates didn't revealed significant phase synchronization neither for the PSI, nor for the DT, except for the DT in one participant and this in all conditions but the Audio Double condition.This confirmed that phase synchronization between the hands and the metronome was not present in the various metronomes conditions and that only a frequency relationship was maintained.2 = 12%, p = .037), ThVel , and DBTz . Moreover, we found that the frequency difference (FSI) affected the variability of DBTz in the case of the antiphase audio-tactile metronome  more than in the other conditions Click here for additional data file.