Human clinical uses for diagnostic and image-guided surgery are also emerging. In comparison to low-molecular weight organic dyes, the use of fluorescent nanoprobes can improve both the signal sensitivity  and the fluorescence biodistribution (passive “nano” uptake in tumours for instance). A wide range of fluorescent nanoprobes have been designed and tested in preclinical studies for the last few years. They will be reviewed and discussed considering the obstacles that need to be overcome for their potential everyday use in clinics. The conjugation of fluorescence imaging with the benefits of nanotechnology should open the way to new medical applications in the near future.With the fast development, in the last ten years, of a large choice of set-ups dedicated to routine  From the first anatomical charts to the most recent development of Magnetic Resonance Imaging (MRI) or Positron Emission Tomography (PET), setting into images what is normally unseen within living systems has been a major issue for the understanding of biological processes ,2,3. Witin vitro and superficial in vivo imaging applications. However, fluorescence imaging using the near infrared range is also now routinely used for whole body imaging of small animals in preclinical studies . . 50 ≈ 30articles c [24,142Another strategy is to use as fluorescent dye nanocarriers natural lipoproteins present in the blood stream, especially high density lipoproteins (HDLs) and low density lipoprotein (LDLs) ,146. Lipvβ3 integrins [in vivo administration.Phospholipid-PEG micelles were shown to improve ICG optical properties and prolong up to a few weeks its stability in aqueous buffer ,152. Thentegrins . However18 ester dye were used for near infrared optical imaging of cultured macrophages and of inflammatory process like oedema in an in vivo mouse model [Liposomes labelled with the DY-676-Cse model . ICG wasse model ,155, artse model  or lymphse model . Porphysse model . 50 values as high as 0.1 to 1 mg/mL of lipids for liposomes or solid lipid nanoparticles [To conclude on lipid-based fluorescent nanoprobes, specific features seem to be associated to these nanostructures. Indeed, lipid-based nanocarriers have been reported to display strong affinity for lymphatic channels and lymph nodes ,138,142,articles ,141. TakDuring the last decades, advances in technology  have enabled the development of high performance instrumentation for near-infrared fluorescence imaging. This imaging modality has rapidly been propelled toward clinical applications, since the near-infrared region is the best suitable optical window for body in-depth scanning. The use of the currently available near-infrared dyes, especially FDA-approved ICG, has been fully revisited by extending the field of their clinical applications. Due to the depth issue (photon penetration into tissues), near-infrared fluorescence imaging applications have been restricted to superficial tissues, or deep tissues in intraoperative context. For instance, clinical trials are presently ongoing on the accurate identification and resection of the sentinel lymph node, using fluorescence guided surgery. In fine, the choice of a fluorescent nanoprobe dedicated to a specific medical application will be discussed on the basis of toxicity/adverse effects at the dose to be injected for good image contrast. The same criterion will dictate the choice between small organic dye and nanometer size tracer. Certainly, several nanoprobes of different chemical nature will come to market for different applications in the near future.In the meantime, nanotechnologies have opened exciting new avenues in the quest of more photostable and highly bright near-infrared tracers, more performing than classical organic dyes in terms of optical properties. The use of nanostructures in comparison to small organic dyes is mainly driven by their different and tunable biodistribution that can be tuned according to article size, shape, charge and flexibility. The wide range of nanostructures explored for the design of efficient nanoprobes is impressive. Quantum dots present several strong physicochemical advantages for this purpose, but their short term translation toward clinics seems impeded by the toxicity issue still in debate for their use in patients. Another encouraging way relies on the entrapment of organic near-infrared dyes into toxic element-free nanoparticles, especially those developed in pharmaceutical science for drug delivery. These safe nanoparticles, especially polymer- or lipid- based carriers, allow the protection of the near-infrared dyes against chemical and/or biological degradation, improve their photophysical properties, and can be targeted for reaching specific diseased cells, increasing thus the fluorescence signal-over-noise ratio. The development of such interesting smart nanoprobes usually relies on interdisciplinary teams in basic sciences in fields ranging from physics to biology, in close environment to clinicians. Their future translation to clinics depends on the hardiness of their production processes in Good Manufacturing Practice (GMP) conditions, their compelling safety, and the benefit they may offer for human health in indicated clinical applications . Taking all together the aforementioned elements, there is no doubt that new clinical uses of fluorescent nanoprobes will emerge in the next few years.


Most existing person re-identification methods focus on matching still person images across non-overlapping camera views. Despite their excellent performance in some circumstances, these methods still suffer from occlusion and the changes of pose, viewpoint or lighting. Video-based re-id is a natural way to overcome these problems, by exploiting space–time information from videos. One of the most challenging problems in video-based person re-identification is temporal alignment, in addition to spatial alignment. To address the problem, we propose an effective superpixel-based temporally aligned representation for video-based person re-identification, which represents a video sequence only using one walking cycle. Particularly, we first build a candidate set of walking cycles by extracting motion information at superpixel level, which is more robust than that at the pixel level. Then, from the candidate set, we propose an effective criterion to select the walking cycle most matching the intrinsic periodicity property of walking persons. Finally, we propose a temporally aligned pooling scheme to describe the video data in the selected walking cycle. In addition, to characterize the individual still images in the cycle, we propose a superpixel-based representation to improve spatial alignment. Extensive experimental results on three public datasets demonstrate the effectiveness of the proposed method compared with the state-of-the-art approaches. Person re-identification (re-id), as an important technique to automatically match a specific person in a non-overlapping multi-camera network, has been widely used in many applications, such as surveillance ,2, forenExisting works on feature representation mostly rely on still person images across non-overlapping camera views. These works can further be divided into two groups according to the number of images they use, namely, single-shot re-id and multiple-shot re-id. Single-shot re-id methods use one single image to model the person, which are evidently limited in that they do not make full use of the information. Because, in real-world applications (like surveillance), there is usually a sequence of images available for a person in each camera view. As a consequence, the single-shot methods often suffer from some practical challenging factors, like occlusion, and pose, viewpoint or lighting changes. Multiple-shot re-id methods ,27,28,29To tackle the limitations mentioned above, some authors recently advocate video-based re-id, which can usually achieve better performance by exploiting the abundant space–time information in feature representation. Compared to still-image-based re-id, where only spatial alignment needs to be considered since the primary challenge in feature representation is to achieve robustness to viewpoint changes, one fundamental but challenging problem in video-based re-id is temporal alignment. Considering its significance, temporal alignment has been studied in very recent literature ,31, bothTo this end, we present a superpixel-based Temporally Aligned Representation (STAR) method to address the temporal alignment problem for video-based person re-identification. More precisely, we first extract motion information from the input sequence by tracking the superpixel of the lowest portions of human, to build a candidate set of walking cycles. Second, on the assumption that there is a whole walking cycle in the video of each person, we select the “best” walking cycle to perform temporal alignment across videos. Finally, to extract the representation of the selected walking cycle of a video, we propose a superpixel-based representation for each single image, and a walking cycle based temporally aligned pooling method.The preliminary version of portions of this paper has been published in . Compare(1)We propose a robust temporal alignment method for video-based person re-id, which is featured by the superpixel-based motion information extraction, the effective criterion for candidate walking cycles, and the use of only the best walking cycle to build the appearance representation.(2)Superpixel-based support regions and person masks are introduced to still image representation, so as to improve spatial alignment and thereby alleviate the undesired effects of the background.(3)The proposed method, performs favorably against the state-of-the-art methods, even deep learning-based ones.To sum up, the contributions of this paper are as follows:The rest of this paper is organized as follows. Related work is reviewed in Person re-id has attracted much attention in recent years, and we first point the readers to some literature surveys on this topic ,5,6,7,33In real video surveillance applications, the information for a person is recorded by a video or an image sequence, rather than a single image. Person re-id based on video is an effective way, due to abundant information in videos, such as underlying dynamic information. Some works have validated the superiority of video-based re-id methods ,49,50,51The direct way to use the video information is the multiple-shot re-id methods ,28,29,52Temporal alignment. Temporal alignment has been demonstrated to be able to lead to a robust video-based representation in the context of gait recognition . Some reAlthough the methods ,31 have To address the aforementioned problems, this paper proposes a superpixel based temporal alignment method, by first extracting the superpixels on lowest portions of human in the first frame, and then tracking them to obtain the curves of their horizontal displacements, finally selecting the “best” cycle in the curves. Note that only one cycle is used for person representation, to address the problem of redundancy and cluttered background and occlusions. Our work is partially inspired by two aforementioned video-based re-id approaches ,31. Howe(1)We adopt a superpixel-based strategy to extract the walking cycles of walking person, i.e., by tracking the superpixels of the lowest portions of human , instead of relying on the motion information of all the pixels of the lower body as in previous approaches ,31, whic(2)We propose to use only the best walking cycle for temporal alignment, rather than using all the walking cycles. An effective criterion based on the intrinsic periodicity property of walking persons is proposed to select the best walking cycle from the motion information of all the superpixels. The motion information of a superpixel matches the human walking pattern, to some extent means the superpixel lies on person, and not be occluded.(3)Based on our temporal alignment, we introduce a novel temporally aligned pooling method to establish the final feature representation. More specifically, we take superpixels as the region supports to characterize the individual still images. Meanwhile, we then utilize person masks to enhance the robustness further.Spatial–temporal representation. The gait based feature is the reliable information for person re-id . HoweverConsidering that most 3D representations are extensions to some widely used 2D descriptors, this paper proposes a simple framework to build a 3D representation, by combining single image based representations and temporally aligned pooling. Another reason is that lots of successful single image based representations have been proposed for person re-id, which are person re-id specific, such as LOcal Maximal Occurrence representation (LOMO) , GaussiaMetric learning. Learning a reliable metric for video matching is another important factor for the video-based re-id ,56,68,69If we extract a fixed-length representation for a video sequence, the metric learning methods for video-based re-id is same with that for single-image-based re-id. Thus, the single-image-based metric learning methods can be used directly, such as KISSME in ,31. In tAccording to its characteristics, we list three main steps in the video-based re-id methods: temporal alignment, spatial–temporal representation, and metric learning. Many works related to these steps have been proposed recently:It is stressed that deep networks based frameworks do not follow the steps mentioned above, but train an end-to-end neural network architecture ,72,73,74This paper focuses on appearance representation for video-based person re-id. In this section, we introduce the proposed superpixel-based temporally aligned representation by (1) extracting motion information based on superpixel tracking , (2) selWe propose a superpixel based motion information extraction, which ought to be more robust than pixel based methods, because (1) it is based on superpixels, dynamic information extraction is robust to noise caused by some individual pixels, while pixels based on methods like FEP suffer from this; (2) to alleviate the effect of occlusions and cluttered background, we utilize the local superpixel to extract motion information, and then select the “best” cycle in the curves of all the superpixels. Note that, although the superpixels may also be on the background, whose curves of motion information are quite easy to be distinguished from that on person, as discussed in T frames, we extract the motion information, as illustrated in N superpixels Given a video sequence N superpixel to extract the motion information. For the jth superpixel V, resulting in a set of superpixel T elements, where tth frame. Note that, although we use a set to describe the superpixels, it is ordered, and the superpixel in the sets are in the same order as the frames in the corresponding video.Then we track all the t, we extract x. In this paper, we use the color feature, i.e., HSV histogram, to represent the superpixels. jth superpixel At frame It is worth noting that we track all the superpixel on the lowest portion of an image, without segmenting the lowest portions of human out of the background. The main reasons are two-fold: (1) foreground segmentation is not dependable since it is difficult due to noise, occlusions, and low-resolution ; (2) the motion information of the superpixels in the background actually is easy to eliminate, since the motion information of the superpixels from the foreground and from the background in the lowest portion of the frame is quite different. N superpixels, we are trying to select the “best” walking cycle x, y and z directions. That is consistent with the biomechanics of gait [To address the problem of abundance and noise caused by cluttered background and occlusion, we use only one walk cycle of frames to represent a video sequence. Therefore, after obtaining the redundant motion information   of gait . (2) TheBy modeling the horizontal displacements of feet as a sinusoid, we propose an effective criterion to select the “best” walking cycle. We expect the criterion has two characteristics: (1) it should be a complete walking cycle  since it covers the entire dynamic information and variety of poses and shapes, and (2) it should be quite similar to sinusoid, namely, with less noise caused by cluttered background and occlusion. That is, we search the complete candidate walking cycles from the curves of the horizontal displacements according to the prior of walking persons. Then we evaluate how good a candidate walking cycle is, by measuring its fit error to a sinusoid.c is the location of the horizontal center line, i.e., W is the width of the image. We set the bounds to filter the extreme points on the smooth curve, and alleviate the influence of noise and occlusions.Specifically, we first try to find candidate walking cycles from the motion information curves based on extreme points, as shown in K extreme points and by k-th extreme point We denote by jth superpixel is, we fit its positions W is the width of the image. The final selected walking cycle To evaluate how “good” a candidate cycle jth superpixel We also present an example to visually illustrate our algorithm in As mentioned in O is bigger than the threshold To sum up, we proposed a superpixel-based LOMO (SPLOMO) representation for still images, as shown in Given the selected walking cycle M segments M segments in the walking cycle. We describe the phase m as Note that the frame numbers of walk cycles in different video sequences are usually different, which is not convenient to learn a metric. An alternative modality is multi-versus-multi (MvsM), in which there is a group of multiple exemplars for each person in the gallery and group of multiple images of each person in the probe set. However, temporal information of a video sequence is missing in the MvsM method, while it is quite important in video-based re-id. To address this problem, we propose to use temporally aligned pooling method to normalize the representations of all the frames, according to the intrinsic periodicity property. That is, we perform temporally aligned pooling according to the sinusoid corresponding to the walking cycle. Specifically, we equally divide the sinusoid into https://github.com/chaunceygao/STAR) and compare it to other state-of-the-art approaches on three public video-based re-id datasets.In this section, we validate the proposed STAR method   is used ing SLIC , with thhttp://mmlab.ie.cuhk.edu.hk/projects/luoWTiccv2013DDN/index.html) [In SPLOMO, we extract person masks using DDN (The code is available on ex.html) . Figure ex.html) ).In this section, we report the comparison results of STAR with the existing state-of-the-art video-based person re-id approaches on iLIDS-VID, PRID 2011 and MARS datasets. Three groups of the approaches are compared, as shown in We also report the results of TAPR, which is the preliminary version of our method. The main difference of TAPR and STAR is that TAPR describes each frame with LOMO feature, while STAR describes it with a SPLOMO feature. As mentioned above, SPLOMO improves LOMO by introducing the constraint of superpixel and person masks for better spatial alignment. The comparison results of these two methods show that our proposed superpixel based representation improve the performance of LOMO distinctly, especially on the iLIDS-VID dataset. More specifically, the rank-1 identification rate is 67.5% for STAR on the iLIDS-VID dataset, while 55.0% for TAPR. This validates the effectiveness of the SPLOMO representation on spatial alignment.It is worth noting that we achieve the best performance on both iLIDS-VID dataset and MARS dataset, and a comparable result on the PRID 2011 dataset. We believe this is because the iLIDS-VID and MARS datasets have more occlusions and cluttered background than the PRID 2011 dataset. And the proposed STAR can excavate the “best” walking cycle to reduce their effect and achieve more accurate temporal alignment. Moreover, STAR uses the person masks to avoid the effect of the background, and uses superpixel based representation to achieve better spatial alignment. This means (1) accurate spatial and temporal alignment is quite essential in the video-based re-identification, especially when the scenes are complex; (2) our method has great abilities in video temporal alignment even with complex scenes.Frame#4-28 is partially occluded, and the walking cycles Frame#29-52 and Frame#40-66 both have cluttered background, while the walking cycle Frame#14-38, which is the selected one, has less noise than others. In Frame#3-25 suffers from occlusion, and the walking cycle Frame#3-25 suffers from occlusion and clutter background, while the “best” one Frame#43-67 has less noise. These examples demonstrate that our walking cycle method can select the ones with fewer occlusions and cluttered background. We believe that building a representation based on the selected walking cycle can alleviate the effect of occlusions and cluttered background.To alleviate the effect of occlusions and cluttered background, we propose to select the “best” walking cycle (a gait period). To further demonstrate the good performance of the proposed walking cycle selected method, we present two more examples in Frame#26-42 with the green bounding box in Note that the image sequence We evaluate the effects of the mentioned three temporally aligned pooling manners of our STAR algorithm on the iLIDS-VID dataset, i.e., average pooling (STAR_avg), max pooling (STAR_max), and key frame pooling (STAR_key), as shown in Surprisingly, compared with N segments according to the corresponding sinusoid curve. Here we evaluate the effects of the number of segments N, as reported in To devise the temporally aligned representations, we equally divide each walking cycle into We also evaluate the influence of the number of superpixels in We have proposed a novel superpixel-based temporally aligned representation for video-based person re-identification. This representation focuses on both spatial and temporal alignment problems in video-based representations. To achieve temporal alignment, we select a video fragment of a walking cycle and describe the video fragment using temporally aligned pooling. To further improve spatial alignment, a superpixel is introduced to extract motion information and describe a still image. Unlike most previous video-based representations for re-id that use all the frames to build a spatio–temporal feature, we proposed to use only a “best” walking cycle, to reduce redundant information and simultaneously keep the “best” information. The extensive experiments, conducted on iLIDS-VID, PRID 2011 and MARS datasets, demonstrate that our method outperforms the state-of-the-art approaches.


Living in social hierarchies requires individuals to adapt their behavior and physiology. We have previously shown that male mice living in groups of 12 form linear and stable hierarchies with alpha males producing the highest daily level of major urinary proteins and urine. These findings suggest that maintaining alpha status in a social group requires higher food and water intake to generate energetic resources and produce more urine. To investigate whether social status affects eating and drinking behaviors, we measured the frequency of these behaviors in each individual mouse living in a social hierarchy with non-stop video recording for 24 h following the initiation of group housing and after social ranks were stabilized. We show alpha males eat and drink most frequently among all individuals in the hierarchy and had reduced quiescence of foraging both at the start of social housing and after hierarchies were established. Subdominants displayed a similar pattern of behavior following hierarchy formation relative to subordinates. The association strength of foraging behavior was negatively associated with that of agonistic behavior corrected for gregariousness (HWIG), suggesting animals modify foraging behavior to avoid others they engaged with aggressively. Overall, this study provides evidence that animals with different social status adapt their eating and drinking behaviors according to their physiological needs and current social environment. Social dominance hierarchies emerge as animals measure the competitive ability of others through social interactions and individuals learn to consistently yield towards relatively dominant individuals . One of ad libitum  and housed in groups of three for 2 weeks in standard sized cages with ad libitum standard chow and water. All mice were individually marked by dying their fur with nontoxic animal markers . The 156 mice were assigned into 13 distinct groups of 12 males. These social groups were part of several different ongoing studies in our laboratory with the aim of analyzing blood and brain tissue post-mortem. The purpose of this study was to assess how feeding and drinking behavior varies with social status across hierarchy formation. Therefore, we acquired 24-hour observations of feeding and drinking behavior for each of the social groups (described below). Animals were undisturbed throughout the experiment.2 = 3 floor ×150 cm ×80 cm) and a lower level with five nest boxes filled with pine bedding  connected by tubes. The total surface of a vivarium is approximately 62,295 cm2, providing 5,191 cm2 per mouse. Standard chow and water were provided ad libitum from two locations on the top shelf of the vivarium. All animals either had no previous experience with any other animal in the group or had been previously housed with only one other male in the social group. Subjects were housed with constant temperature (22–23 °C) and humidity (30–50%) and a 12/12 light/dark cycle with white light (light cycle) on at 2,400 h and red lights (dark cycle) on at 1,200 h. We observed if any animal exhibited a sign of pain or injury every day. All procedures were conducted with approval from the Columbia University Institutional Animal Care and Use Committee .On the day of group-housing 12 mice were weighed and placed into custom-built vivaria , and conAnimals were housed in groups for up to 27 days . An average of 1.70 h of daily behavior observations were undertaken on each group to determine the social hierarchy. Observations always occurred in the dark phase of the light cycle. Trained observers recorded all occurrences of fighting, chasing, mounting, subordinate posture and induced-flee behaviors and the identity of the dominant and subordinate individuals in each interaction  from five cohorts, recording from the time that animals were put into the vivarium. Eating and drinking in stable social hierarchies (Stable) were recorded from 11 separate cohorts between days 6 and 22. By sampling across a range of days we were able to assess if the time since group formation also affected feeding and drinking behavior. A total of three cohorts were videoed for feeding and drinking behavior on both Day 1 and post Day 6 (Stable). We controlled for this using cohort-ID as a random effect in all models where appropriate. We have previously demonstrated that all hierarchies become stable from Day 4 or 5 onwards . We furtp < 0.001)  .All statistical analyses were undertaken in R v. 3.4.3 . The stah-value and triangle transitivity (ttri), and directional consistency (DC) and associated p-values derived from 10,000 Monte-Carlo randomizations  sampling using the MCMCglmm R package . MCMC isx = total number of minute bins where A and B were foraging (eating or drinking) at the identical dispenser location, yA = total number of minute bins with only A identified, yB = total number of minute bins with only B identified, yAB = total number of minute bins where A and B are identified from different locations. As mice differ in their tendency to associate aggressively (gregariousness) across social ranks, we calculated HWIG  as described in (Within stable (post Day 6) cohorts we measured the association strength of foraging behavior by calculating the simple ratio association index (SRI) for each of 726 dyads  . Brieflyribed in  as a mearibed in  to fit mh′ mean = 0.78, interquartile range (IQR) = [0.67–0.81], all p < 0.001; ttri = 0.87 [0.83–0.93], all p < 0.001; DC = 0.86 [0.84–0.92], all p < 0.001). All 11 cohorts videoed after Day 6 had formed a stable linear hierarchy by the day of video recording . In 9/11 groups the alpha male on the day of video recording was the same alpha male at the end of group housing. Although in the other two groups the final alpha male was ranked 2 and 3 respectively on the days of video recording, these two alpha males maintained their alpha status for at least 13 days after the day of video recording (Cohort A and E in p > 0.68).All cohorts formed a linear hierarchy over the housing period   = , pMCMC =0.099, We further examined differences in eating and drinking frequency among three social status groups: alpha , subdominant  and subordinate  see . On Day There were no significant differences in average eating or drinking bout duration across all ranks  and 14% of mice in established hierarchies  ate and drank more during the light phase compared to the dark phase . SubdomiWe also analyzed the longest duration of inactivity in eating/drinking behavior for each mouse . For 82%In each vivarium, mice could eat and drink from one of two hoppers. One was placed in the top right of the vivarium and the other in the top left. We used a binomial test to see if mice showed a location preference between the two dispensers. Out of 60 mice observed on Day 1, 45 mice showed significant preferences for one particular food/water location; seven animals preferred the left food hopper and 38 animals preferred the right one. Among the five cohorts observed on Day 1, the alpha males from three cohorts showed a significant location preference. Eighteen of the 27 non-alpha males in those three cohorts preferred the same location that the alphas preferred, and nine males chose to visit the other location more often. For the 11 cohorts we observed after the social hierarchies were stabilized, 96 out of 132 mice showed a location preference . The alpha males of seven stable cohorts significantly preferred one specific location, and 42 out of 60 non-alpha males in those seven cohorts preferred the same location as their respective alpha males did . It is cWhether animals avoid associating with the alpha male in their groups while eating or drinking was more completely addressed by comparing the mean difference of association indices of eating and drinking behaviors at feeding/drinking locations between alpha-other and other-other relationships. Overall, associations were very low and individuals associated with the alpha male at a similar rate as they did with other males  and on all days measured after hierarchies were established. Although we were not able to directly measure the amount of food and water each mouse consumed, we show that the durations of individual eating and drinking bouts across light phases is not different across social ranks, suggesting that the frequency of eating and drinking is a reliable measure of the amount of food and water each animal consumed. Dominant animals in a social hierarchy, especially the alpha male of a group, initiate and engage in a significantly higher number of aggressive interactions than relatively subordinate individuals  requirinTo our knowledge, this is the first time that the drinking frequency of all mice living in large social housing has been recorded with non-stop recording for a full light/dark cycle while evaluating all individuals’ social status. We confirmed our hypothesis that more dominant individuals in the social hierarchy drink water more frequently, as predicted from our previous finding that individuals with higher social ranks produced a higher volume of urine daily . Our finAnother possible explanation for the finding that subordinate mice eat and drink less frequently is that they experience higher levels of social stress leading to appetite loss . We haveWe also show that mice visit food and water dispensers more frequently during the dark phase compared to the light phase of the light cycle. This is consistent with previous findings that mice are more active and intake more food during the ‘active’ dark phase . InteresIn this study we demonstrate how individual social status associates with feeding and drinking behavior in social hierarchies of male mice. In combination with our lab’s previous findings showing the dramatic increase in MUP production and daily urination volume by alpha males, we propose that maintaining alpha status in social groups is metabolically expensive and requires dominant male mice to consume more food and increase water intake. This dynamically changes their temporal patterning of foraging behavior and may influence the behavioral patterns of other individuals in their social group. Additionally, we also show that outbred CD-1 mice are able to flexibly adapt their foraging behavior based on past agonistic interactions suggesting a degree of social competence. We believe these current results lay a basis for future studies examining the neurobiological and physiological mechanisms connecting perception of social status and critical physiological adaptions that occur during the establishment and maintenance of social hierarchies.10.7717/peerj.5617/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.5617/supp-2Supplemental Information 2Click here for additional data file.


WAF1/CIP1 (p21) plays critical roles in cell‐cycle regulation and DNA repair and is transcriptionally regulated through p53‐dependent or ‐independent pathways. Bioinformatic analysis predicated one stress‐response element (STRE) implicated in single nucleotide polymorphism (SNP) rs2395655 of the p21 promoter. Here, we investigated the transcriptional regulatory function of rs2395655 variant genotype and analyzed its associations with the p21 expression and clinical outcomes in esophageal squamous cell carcinoma (ESCC) patients. Luciferase assay results showed significantly increased transcriptional activity of the rs2395655 G allele‐containing p21 promoter compared with rs2395655 A allele‐containing counterpart, especially in ESCC cells with ectopic LEDGF/p75 expression. Furthermore electrophoretic mobility shift assay using the rs2395655 G or A allele‐containing probe and chromatin immunoprecipitation assay with specific anti‐LEDGF/p75 antibody indicated the potential binding activity of LEDGF/p75 with the STRE element implicated in rs2395655 G allele of the p21 promoter. Subsequent specific RNA interference‐mediated depletion or ectopic expression of LEDGF/p75 caused obviously down‐ or up‐regulated expression of p21 mRNA in ESCC cells harboring rs2395655 GG genotype but not cells with rs2395655 AA genotype. Furthermore, rs2395655 GG genotype carriers showed significantly elevated p21 protein expression and conferred survival advantage in both univariate and multivariate analyses in total 218 ESCC patients. Our findings suggest that LEDGF/p75 regulates the p21 expression in ESCC cells through interacting with STRE element implicated in polymorphism rs2395655 and the elevated p21 protein expression and rs2395655GG genotype may serve as positive prognostic factors for ESCC patients.p21 Esophageal squamous cell carcinoma (ESCC) constitutes 80% of esophageal cancer and the majority of ESCC cases occur in Asia, especially in north central China.p21WAF1/CIP1 p21, AF 497972) is an essential mediator in the DNA damage response, by inducing cell cycle arrest, direct inhibition of DNA replication, as well as by regulating cell apoptosis. p21 interacts with cyclin/cyclin‐dependent kinase (CDK) complex and functions to negatively control cell cycle.97972 is cis‐elements corresponding to these external and internal factors.cis‐elements into cellular decisions leading to proliferation or cell cycle arrest.Transcriptional regulation is the key mechanism in control of the p21 expression. The activation of p21 promoter is regulated both by transcription factor p53 in response to DNA damage and by extracellular growth stimuli in a p53‐independent mechanism and the p21 promoter harbors several http://www.cbrc.jp/research/db/TFSEARCH.html) and predicted that one cis‐regulatory element In this study, we performed bioinformatic analysis using the TFSEARCH program  and 5′‐GGAAGATCTGAAACACCTGTGAACGCAGCAC‐3′ (reverse). The polymerase chain reaction (PCR) product was ligated into pGL3‐Basic vector  and the resultant reporter construct was used as template to generate three other constructs containing −2119A/−809G, −2119C/−809A, and −2119A/−809A haplotypes, respectively, by using site‐specific mutagenesis. Expression vector containing the full coding domain sequence of LEDGF/p75 gene was constructed using the pcDNA3.0 plasmid . All constructs were restriction mapped and sequenced. Two small interference RNAs (siRNAs) targeting nucleotides 1428 to 1448 and nucleotides 1340 to 1360 of human LEDGF/p75 mRNA (NM_033222.3) were synthesized and named as si‐p75‐1 and ‐2, respectively.2.22 at 37°C. LEDGF/p75 expression vector and si‐p75 transfection were performed as described.4 cells/well). The next day, 0.8 μg of expression vector or si‐p75 was mixed with 2 μL Lipofectamine 2000 (Invitrogen) plus 50 μL Opt‐MEM medium (Invitrogen) for 20 minutes and then added to cells. For ectopic LEDGF/p75 expression and siRNA transfection, the empty vector (pcDNA3.0) and mismatched siRNA were used as controls, respectively. After transfection for 24 hours, cellular proteins were isolated for further analysis.Four kinds of human ESCC‐derived cell lines  were kindly provided by Dr. Chen KN . Cells were cultured in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin stock in a humidified atmosphere of 5% CO2.3EC‐109 and KYSE150 cells were seeded into 24‐well plates. Twenty‐four hours later, cells were cotransfected with 0.4 μg of one reporter construct containing a p21 promoter haplotype, 2 ng of pRL‐SV40, and 0.4 μg of LEDGF/p75 expression vector or empty vector (pcDNA3.0) using Lipofectamine 2000 (Invitrogen). Luciferase activity was analyzed 48 hr after transfection using the Dual‐Luciferase Reporter Assay System (Promega). The pRL‐SV40 co‐transfected with pGL3‐Basic vector and pcDNA3.0 empty vector was used as control. The value of each report construct was calculated as the mean ± SD from three independent experiments each performed in duplicate.2.42, 50 ng/μL Poly(dI:dC), and 0.05% NP‐40. The resulting protein‐DNA complexes were subjected to electrophoresis on a 6% native PAGE, transferred to a positively charged nylon membrane , and then cross‐linked using a GS Gene Linker UV chamber . To demonstrate the specificity of protein‐DNA complex formation, an anti‐LEDGF/p75 antibody (C‐16)  or a 400‐fold molar excess of cold probe was added to the binding reaction. Biotin‐labeled DNA was detected using stabilized streptavidin/horseradish peroxidase conjugate (Pierce).Nuclear extracts were prepared from KYSE150 cells with NE‐PER Nuclear and Cytoplasmic Extraction Reagents according to the manufacturer's instructions . Synthetic double‐stranded oligonucleotides 5′‐GCAACCACAGGGATTTCTTCTGTTC‐3′ and 5′‐GCAACCACAGGGGTTTCTTCTGTTC‐3′ corresponding respectively to rs2395655 G and A alleles were labeled with biotin and the binding reactions were performed using the LightShift Chemiluminescent EMSA Kit (Pierce). For each gel shift reaction, 20 fmol of biotin‐labeled probe was incubated with 4 μg of nuclear extracts for 20 minutes at room temperature in a 20 μL mixture containing 1 × binding buffer, 2.5% glycerol, 5 mmol/L MgCl2.5Chromatin immunoprecipitation was performed using EZ‐Magna ChIP TMA Chromatin Immunoprecipitation Kit  following the manufacturer's instructions. Formaldehyde cross‐linked chromatin obtained from EC‐109 and KYSE410 cells was sonicated to generate DNA fragments from 200 bp to 1000 bp. Anti‐LEDGF/p75 antibody (C‐16) (Santa Cruz) was used to precipitate LEDGF/p75. ChIP sample (5% of input) was used as a positive control and IgG (Santa Cruz) as a negative control. Oligonucleotide primers used to amplify genomic DNA for ChIP are shown in Table 2.6g for 25 minutes. The total protein concentration was quantified utilizing a BCA protein assay kit and equal amounts of proteins (10‐20 μg/lane) were separated on 10%‐12% SDS‐PAGE gels and electrotransferred to PVDF membranes. The membranes were then blocked with 5% nonfat milk and probed with specific primary antibodies: anti‐LEDGF/p75  or anti‐Actin (Santa Cruz) (1:1000 to 1:5000 dilution), respectively. After washing with 0.2% PBS‐T buffer four times, membranes were incubated with horseradish peroxidase‐conjugated secondary antibody and visualized using the enhanced chemiluminescence system, ECL .Cells were lysed on ice for 25 minutes in lysis buffer  supplemented with protease inhibitors and the solution was cleared by centrifugation at 14 000 2.72, 0.01% gelatin, 200 pmol for each primer, 2 U Taq DNA polymerase (Promega), and 2 μL of sample cDNA. The resulting fragments were subjected to electrophoresis on a 1%‐2% agarose gel and visualized with ethidium bromide staining. All PCR reactions were repeated with reproducible results.Total cellular RNA was isolated with Trizol reagent  according to the manufacturer's instruction. Approximately 2 μg of total RNA from each cell line was digested with DNase I to remove DNA contamination and reverse transcribed into cDNA using the Superscript III System (Invitrogen). To examine the expression levels of LEDGF/p75 and p21 in ESCC cells, specific primer sets were designed and β‐actin was used as internal control. The primer sets, annealing temperature for amplification, and the length of PCR products are listed in Table 2.8Total 218 paraffin‐embedded ESCC tissues were retrieved from the Pathology Department of Peking University Cancer Hospital & Institute from July 2003 to December 2007. The enrolled patients involved 156 men and 62 women, ages 38 to 79 years , with stage I (n = 19), IIa (n = 79), IIb (n = 59), and III (n = 61) diseases according to the TNM system on the basis of AJCC classification.2.9Formalin‐fixed and paraffin‐embedded tumor sections were histopathologically reviewed by two trained pathologists and the cancer tissues were separated using manual microdissection. Tissues were deparaffinized in xylene and then incubated overnight at 56°C in 50 μL of the digestion buffer containing 10 mmol/L Tris, pH 7.6, 100 mmol/L NaCl, 10 mmol/L EDTA, pH 8.0, 0.5% Tween 20, and 200 μg/mL proteinase K. The next day, proteinase K was inactivated by incubation of the samples at 100°C for 10 minutes. DNA samples were stored at −80°C until analysis. The rs2395655 variant genotype of the p21 promoter was determined by PCR direct sequencing and the primer sets are listed in Table 2.102O2 solution. The slides were counterstained with 5% hematoxylin, and then examined by light microscopy. Sections without primary antibody treatment were used as negative control. Immunohistochemical evaluation of the p21 protein expression in ESCC specimens was based on the intensity and extent of nuclear reactivity. Moderate to strong p21 nuclear staining in ≥10% of tumor cells was defined as positive result. All ESCC sections were histopathologically reviewed by two trained pathologists.All tissues were stained using S‐P immunohistochemical method. Briefly, the slides were dewaxed in xylene, rehydrated in graded alcohols, and then treated with PBS containing 3% hydrogen dioxide to block endogenous peroxidase activity. After preincubating in 10% goat serum for nonspecific binding block, slides were then incubated overnight at 4°C with mouse anti‐human p21 protein . After rinsing with 0.1% PBS‐T, sections were subsequently incubated with biotin‐conjugated IgG (Santa Cruz) with 1:10 000 dilution for 15 minutes at room temperature, followed by incubation with streptavidin‐peroxidase conjugate for 15 minutes. The signals were developed with DAB‐H2.11t test. The associations of clinicopathologic characteristics and genetic variant with the p21 protein expression were tested by χ2 test. Disease‐free survival was defined as survival without the development of local recurrence or distance metastases. Univariate survival analysis was carried out by Kaplan–Meier method using log‐rank test. The Cox proportional hazards model was used for multivariate analysis. The variables in the multivariate analysis were age, sex, tumor location, tumor cell differentiation, TNM stage, p21 expression, and rs2395655 variant genotype. Values of P < 0.05 were considered statistically significant.SPSS 15.0 software  was used in determining statistical significance. The continuous variables from different groups were compared using 33.1To evaluate the effects of polymorphisms rs3829963 –2119C/A) and rs2395655 (–809G/A) on transcriptional activity of the p21 promoter, reporter constructs were created with each combination of the two SNPs , comprising nucleotides −810 to −805 of the p21 promoter .The representative ESCC examples with p21 staining positive were shown in Figure 3.5P = 0.001). Notably, ESCC patients carrying rs2395655 GG genotype showed better postoperative outcome than patients with rs2395655 AA or AG genotype  , as shown in Table Univariate analysis using the log‐rank test revealed that the p21 protein expression and rs2395655 variant genotype were significantly associated with disease‐free survival time of ESCC patients. As shown in Figure 4cis‐acting elements in the p21 promoter region and regulate p21 gene transcription.p21 is a principal mediator of cell cycle arrest in response to DNA damage and its expression is regulated by a complex cellular signaling pathway in which many nuclear proteins, including p53, E2F, Ap‐1, Ap‐2, c‐Jun, et al., interact with cis‐acting elements, namely, STRE and HSE (heat shock element), located in the promoter region of numerous genes, including antioxidant protein 2, Hsp27, alphaB‐crystallin, and VEGF‐C.LEDGF/p75 is a survival factor that enhances growth and resistance to cell death induced by various environmental stress signals and regulates downstream response pathways by enhancing expression through binding to specific cis‐regulatory element, that is STRE element, into the p21 promoter region. Reporter constructs encompassing nucleotides −2308 to +204 of the p21 promoter with each combination of the above two SNPs were established for luciferase assay and the results showed that polymorphism rs2395655 but not rs3829963 influenced the p21 promoter activity in the context of the 2‐site haplotypes. Moreover, the p21 promoter containing rs2395655 G allele displayed significantly higher transcriptional activity than A allele‐containing counterpart, especially with ectopic LEDGF/p75 expression in ESCC cells. Furthermore, gel shift assay using the rs2395655 G or A allele‐containing probe and chromatin immunoprecipitation assay with specific anti‐LEDGF/p75 antibody indicated the potential binding activity of LEDGF/p75 with the STRE element implicated in rs2395655 G allele of the p21 promoter.In this study, two SNPs, that is rs3829963 (−2119C/A) and rs2395655 (−809G/A), were evaluated for their effects on the p21 transcriptional activity in ESCC cells. Bioinformatic analysis showed that rs2395655 A>G transition introduced one Next, four human ESCC cells with rs2395655 GG or AA genotype were used to examine the potential role of LEDGF/p75 and polymorphism rs2395655 in regulation of transcriptional activity of the p21 promoter. Specific RNA interference‐mediated depletion or ectopic expression of LEDGF/p75 caused obviously down‐ or up‐regulated expression level of the p21 mRNA in cells harboring rs2395655 GG genotype (EC‐109 and KYSE150) but not cells with rs2395655 AA genotype (KYSE410 and KYSE450). Additionally, si‐p75‐2 played a stronger role in down‐regulating the expression levels of both LEDGF/p75 and p21 in comparison with si‐p75‐1. These results were consistent with Singh DK's findings that p21 expression was obviously down‐regulated in LEDGF/p75‐depleted breast cancer cells.P = 0.008). Moreover, the associations of the p21 protein expression and rs2395655 variant genotype with disease‐free survival time were evaluated in this ESCC population. Both univariate analysis and Cox multivariate analysis showed better postoperative outcome for p21‐positive ESCC patients than p21‐negative patients . Our results were consistent with those reports implying the positive prognostic role of elevated p21 expression in ESCC.P = 0.003). Cox multivariate analysis further indicated a positive role of rs2395655 GG genotype in predicting the postoperative outcome of ESCC patients (P = 0.025). These results of survival analysis were supported by the research findings of Ma H and his colleagues that p21 rs2395655 AA genotype was significantly associated with the increased risk of cancer death of non‐small cell lung cancer .Due to the regulatory effect of polymorphism rs2395655 on the p21 promoter activity in ESCC cells, we further performed immunohistochemical evaluation of the p21 protein expression in total 218 ESCC tissues and investigated the association of rs2395655 variant genotype with the p21 protein expression. Chi‐squared test showed significantly elevated p21 protein expression in ESCC tissues with rs2395655 GG genotype than tissues with rs2395655 AA or AG genotype (Taken together, we demonstrated for the first time that LEDGF/p75 binds to STRE element implicated in polymorphism rs2395655 and regulates the p21 gene expression in ESCC cells and tissues. Our results suggested that the elevated p21 protein expression and rs2395655 GG genotype may serve as positive prognostic factors for ESCC patients. These findings further revealed the complicated roles of LEDGF/p75 as a survival factor and a cancer‐related protein, and provided a rationale for ongoing studies aimed at understanding the stress survival activity and the critical roles of LEDGF/p75 in specific human cancers.The authors have no conflict of interest to declare.


Evidence regarding the role of near work in myopia is conflicting. We developed the RangeLife, a device for continuous, objective measurement of working distance. Four devices were built, calibrated, and validated. Then, adult subjects wore the device on weekdays and weekend days, while simultaneously wearing an actigraph device for objective measurements of light exposure and activity. Subjects maintained an activity log and answered a visual activity questionnaire. RangeLife data were downloaded and binned into 0.10 m intervals. Objective diopter hours (dh), a weighted measure of near work, were calculated. Diopter hours for all subjects were significantly higher on weekdays (14.73 ± 4.67 dh) compared to weekends . 94 ± 1.85% of near and intermediate viewing distances were recorded when the subjects were exposed to mesopic and indoor photopic light levels (<1000 lux), and 80.03 ± 2.11% during periods of sedentary physical activity (<320 counts per minute). Subjective reports of time viewing near and intermediate distances significantly overestimated objective measures (p = 0.002). The RangeLife was shown to provide reliable measures of viewing distance, and can be further utilized to understand potential influences of viewing behaviors on refractive error. Potential influences include time outdoors6, near work8, physical activity10, education11, nutrition12 and urbanization13. Several studies have reported an association between myopia and increased time studying and reading17. Rose, et al., reported the highest odds ratio for myopia was for low time spent outdoors and a high time spent performing near work. However, results regarding the influence of near work have been conflicting, with some studies reporting no significant increased risk of myopia with increased near work20. Inconsistent results concerning the role of near work and myopia onset and progression are likely due to the variable and subjective nature by which near work has been assessed. Previous studies evaluating near work in children have traditionally been performed with parent questionnaires, which are subject to recall and parent biases22. In addition, surrogate measures are often used for near work. For example, some studies have used education, intelligence23, and occupation25 as surrogates for near work.Refractive development is regulated by a complex interaction between genetic, environmental, and behavioral factors26; however, diaries are limited by subject compliance. Another technique previously utilized is the experience sampling method30, in which a pager was dispensed to subjects to send alerts throughout the day. Following each page, subjects were asked to complete a self-report about activities at the moment of the page. While this technique provided real-time sampling of activities and was shown to be capable of detecting the proportion of time spent doing near work, only discreet time points were sampled, typically 5–6 times throughout each day, and results depended on the response rate of the subject, averaging approximately 81%28.Besides parent questionnaires, other studies have utilized diaries to quantify near work26. Diopter hours represents a weighted sum of the time spent in particular activities according to the accommodative demand required to perform the task. For example, hours spent reading at a near distance is given more weight than hours spent using a computer at an intermediate distance. However, this metric does not fully describe the complexity of viewing behaviors that may influence eye growth, such as the temporal properties of near viewing. Relevant viewing behaviors that might affect eye growth include the duration of near viewing sessions, intermittent breaks during near viewing, and absolute viewing distance31. Recent studies in children have shown that continuous reading and closer reading distances are associated with myopia32. Studies in animals have shown that experimental myopia is dependent on the temporal pattern of defocus or form deprivation, rather than the total duration of the myopiagenic stimulus34. For example, Napper, et al., showed that brief interruptions of form deprivation with normal vision significantly decreased the magnitude of myopia in chicks34. These findings suggest that reading breaks might be beneficial during prolonged near tasks. The development of a device that provides continuous, objective assessment of viewing distance offers the opportunity to ultimately assess such behaviors to understand their influence on myopia onset and progression in humans.“Diopter hours” is a metric used to quantify near work behaviors, and can be calculated from visual activity questionnaires. Diopter hours weights various near activities and viewing distances to determine a comprehensive value of near work exposure35. Several studies have utilized light, activity, and sleep monitors to begin to understand the influence of these behaviors in refractive development. Recent studies have provided objective evidence that light exposure is related to time of year36, and that myopic children tend to spend less time outdoors than emmetropic children4. Importantly, these studies revealed discrepancies between traditional subjective reports and objective data for both adults and children37.Wearable electronic monitoring devices are being more commonly utilized to objectively and precisely quantify environmental and behavior factors38. The authors used ultrasonic technology mounted on a headband to continuously measure and log viewing distance while subjects read a newspaper for 10 minutes. The results showed that the device was repeatable with high sensitivity, and that high myopes had a shorter working distance than low/non-myopes. Additionally, the authors found that the subject’s self-reported reading distances varied greatly from objective measures. However, the device was not utilized in subjects’ habitual environment and was limited to two 10 minute experimental periods in the lab.One continuously measuring range finding device has been previously utilized to assess reading distance in adult high and low/non-myopesThe development and implementation of wearable devices for continuous measurement of working distance can be used in conjunction with previously validated light exposure and activity monitors to provide a more complete assessment of visual activity relevant to eye growth and myopia development. Here, we employed infrared time-of-flight technology to develop a spectacle frame mounted device, the RangeLife, for continuous measurement of working distance. Our goals were to calibrate and validate the RangeLife device according to known distances and subjects’ activity logs, and to compare objective measures to traditional subjective visual activity questionnaires. Additional goals included assessing light exposure and activity levels during near viewing, and comparing near viewing behaviors in non-myopic and myopic young adults.All components for the RangeLife were acquired from Adafruit, USA. The RangeLife device consists of a time-of-flight distance sensor which uses a class 1 940 nm laser with nanosecond technology (VL53L0X). The time-of-flight principle is a method for measuring the distance between a sensor and an object, based on the time difference between the emission of a signal and its return to the sensor, after being reflected by an object, enabling accurate distance ranging regardless of the object’s surface characteristics. The sensors were controlled by an Arduino microprocessor coupled to a real-time clock (DS3231 Precision RTC) and a micro secure digital (SD) card data logger . Technical specifications for the distance sensor can be found at https://www.adafruit.com/product/3317. Components were connected using the built-in Arduino Inter-Integrated Circuit (I2C) bus. Power was supplied by a rechargeable 2500 mAh lithium polymer battery to provide approximately 16 hours of continuous distance measurements. The sensor is 1 × 1 × 0.2 cm and attaches to the user’s glasses, with a 1 m wire leading to the microprocessor which is housed with the RTC and battery in a plastic enclosure with a belt clip. The device was programmed to collect data continuously at 1 Hz and write onto files on the SD card. Open-source programming libraries for the sensor, clock, and data logger components were used to write running code for the device using the Arduino language. All code and schematics are available on request.Four devices were constructed and used in this study. Each device was tested on one occasion by one observer for known distances from 0.05 to 2.0 m in 0.05 m intervals to assess stability of the measurements and generate a calibration function. For each device, the sensor was placed along a millimeter scale parallel to ground, and directed towards a wall. The device was set to begin recording at 1 Hz, and started at a position 0.05 m from the wall. Every 150 seconds, the device was moved away from the wall by 0.05 m. The recording continued until the device reached 2.0 m. The data were downloaded, and the distances measured by the device were plotted against actual distances from the millimeter scale. Data from all four devices were plotted and linear regression analysis was used to determine a calibration function.Beam diameter refers to the width of the incident laser beam that bounces off of an object. The more narrow the beam diameter, the more precision the device will have to detect small objects in the line of sight. The device was directed towards a wall and projected onto a concentric millimeter scale. The beam diameter was determined for each of the four devices, for distances from 0.1 to 0.5 m in 0.1 m intervals. The observer viewed the beam through an infrared viewer to measure beam diameter at each distance. For 0.6 m and greater, the projected beam on the surface was too faint to detect with the infrared viewer; therefore, beam diameter was interpolated from 0.6 to 1 m. A linear regression was fit to the data, and the beam diameter was calculated in degrees.Subject testing was conducted with the four RangeLife devices described above. Healthy adult subjects ages 22–41 (n = 23) participated in this study. The study was approved by the Committee for Protection of Human Subjects at the University of Houston and was in accordance with the tenets of the Declaration of Helsinki. Subjects provided informed consent after learning the details of the study. All subjects had best corrected visual acuity of 20/20 or better and no ocular disease.39. Refractive status (myopic or non-myopic) was determined by self-report from the questionnaire using an indirect method technique, which has been shown to have reasonable sensitivity and specificity for determining whether a subject is myopic40. Additionally, subjects were drawn from the student and staff population of the University of Houston College of Optometry and were confident in reporting their refractive status. Previously utilized visual activity questionnaires do not take into account the abundant use of hand held devices, such as smartphones and tablets, that has become more prevalent in the past decade. Therefore, we created the UH NEAR survey, adapted not only from near activity questionnaires, such as the Sydney Myopia Study39, but also from public health and psychology questionnaires. Items were created to account for electronic and printed viewing tasks separately, and reduce redundancy and uncertainty between items. For example, video games, an item commonly included in published questionnaires, creates ambiguous results because subjects engage in video games at near (hand held devices), intermediate (computer) and far (television) distances. We attempted to clarify these factors through the UH NEAR survey.Subjects answered a custom activity questionnaire, the University of Houston Near work, Environment, Activity, and Refraction (UH NEAR) survey (see Appendix 1), adapted from previously published visual activity surveysRangeLife devices were fully charged prior to wear, then mounted on the right temple of a spectacle frame directed approximately 4° nasally, so that the beam was aligned at midline at 0.40 m. Devices were mounted to subjects’ habitual spectacle frame, or, if the subject did not wear spectacles, on a provided frame with plano lenses. The wire ran along the right temple, behind the ear, and to an enclosure, measuring 9 × 6 × 4 cm, that held all of the components. The enclosure was attached via a belt clip or placed in the subject’s pocket.36. Time outdoors was defined as minutes per day exposed to greater than 1000 lux4. Following each full day of wear, RangeLife data were downloaded and binned into 0.10 m intervals to calculate the number of minutes per day spent viewing distances from 0.10 to 1.0 m. Distances were categorized from very near viewing (0.1 cm) to “far” viewing  while wearing the RangeLife so that patterns of light exposure and activity could be correlated to viewing distance. Light exposure (lux) and physical activity (counts per minute) were averaged over one minute epochs, as previously describedData are presented as mean ± standard error unless otherwise noted. Calibration and beam diameter data were fit with linear regressions. Subjective diopter hours (dh) were calculated from the questionnaire using Eq. During calibration testing, each of the four devices showed high stability with no noise in the recording. Raw data from one representative device are shown in Fig. Mean subject age was 27.7 ± 5.6 years, with a range of 22–41 . Thirteen subjects were myopic and 10 subjects were non-myopic, with no significant difference in age between refractive error groups (p = 0.17).One subject with mean daily diopter hours of 63.57 dh was identified as an outlier (Tukey outlier test) and not included in data analysis. Subjective diopter hours for all other subjects was greater for weekdays (26.64 ± 9.16 dh) than for weekends . Subjective daily diopter hours were not significantly different between refractive error groups for weekdays (p = 0.27) or weekends (p = 0.93).For objective measures from the RangeLife device, 19 subjects wore the device for two days each, one weekday and one weekend day. Three subjects wore the device for one weekday only, and one subject wore the device for one weekend day only. On average, subjects wore the device for 12.7 ± 2.2 hours on the weekday and 12.4 ± 1.7 hours on the weekend day (p = 0.61). Based on all subjects’ activity logs and objectively measured distance data, activities were correlated to measured distances at corresponding times. Examples activities for each distance are provided in Table Comparisons between objectively measured weekday versus weekend hours were calculated for the 19 subjects who wore the device on both days. Objective diopter hours was greater for weekdays (14.73 ± 4.67 dh) than for weekends . When analyzed by refractive status, objective weekday diopter hours for non-myopic subjects was 12.73 ± 4.99 dh, which was not significantly different than weekend diopter hours, 11.81 ± 3.7 dh (p = 0.34). For myopic subjects, objective weekday diopter hours was 16.4 ± 3.52 dh, which was also not significantly different than weekend diopter hours, 11.97 ± 5.53 (p = 0.10). While myopic subjects tended to have greater diopter hours during the week than non-myopic subjects , the differences did not reach statistical significance (p = 0.06). When weekday and weekend objective diopter hours were weighted for the week . 0.88 ± 0.28% of near to intermediate working distances were detected when the subject was engaged in moderate or vigorous activity (>1048 counts per minute).Near to intermediate working distances (0.1 to <1.0 m) were compared to objective measurements of light exposure and activity, as measured with the Actiwatch. Data for a representative subject are shown in Fig. From the questionnaire, time spent in near and intermediate work was determined by asking subjects to estimate the amount of hours spent viewing a computer, viewing a handheld device, reading printed material, drawing, painting or writing, and playing cards or board games. Subjects reported a mean daily average of 10.34 ± 0.85 hours engaged in near and intermediate work. From the RangeLife device, time spent in near and intermediate viewing was determined by summing the amount of time spent viewing distances from 0.1 to <1.0 m. Objectively measured mean daily time viewing near and intermediate distances was 6.25 ± 0.39 hours. Objective measures were significantly less than subjective reports (p = 0.002), and there were no correlations between subjective reports and objective measures for either weekdays (p = 0.78) or weekends .The results of this study demonstrate the validity and feasibility of continuous, objective assessment of near and intermediate viewing distances using the RangeLife device. We have presented the first objective quantification of viewing distances throughout a day for young adults. We developed a classification scheme for categorizing near to intermediate distances in 0.1 m bins, and established a method for calculating objective diopter hours. These results support further use of the device to assess differences between larger groups of subjects of various ages and refractive status to understand the influence of near work and viewing behavior in myopia. In particular, the device can be used in future studies to assess not only working distance, but also temporal properties of viewing behaviors.et al., reported that age of onset of myopia was related to close up work activity in early childhood, but not close up work as a young adult; subjects were all military conscripts11. Future studies aimed at assessing objectively measured near viewing patterns in younger subjects are imperative in determining the role of near viewing behaviors in refractive development.Here, near and intermediate viewing was assessed during weekdays and weekends to begin to understand how behaviors might vary based on school/work days versus leisure days. In young adult subjects, we found that objective diopter hours were greater for weekdays (14.73 dh) versus weekends (11.91 dh). While there was a trend for myopic subjects to spend more time engaged in near and intermediate viewing than non-myopic subjects, the difference did not reach statistical significance. We speculate that a larger sample size might clarify whether differences in viewing behaviors exists between refractive error groups in young adults. However, it is not unexpected that adult subjects with fairly homogenous occupations (i.e. students and staff at a university) would demonstrate similar behaviors. Saw, 42; however, it remains unclear if viewing behaviors during bright light exposure, such as engaging in near work while outdoors, might contribute to myopia development. Future studies making use of objective measures for these multifactorial components will help to elucidate contributing behaviors to myopia, and may allow researchers and clinicians to suggest modifiable behaviors that will reduce the onset and progression of myopia.By analyzing multiple objective measures of behavior, including working distance using the RangeLife and light exposure and physical activity using an Actiwatch, we can begin to tease apart specific behaviors that may influence eye growth. We found that the majority of near and intermediate work was carried out while the subjects were exposed to mesopic (<10 lux) and indoor photopic (<1000 lux) illuminance. Additionally, subjects were primarily engaged in a sedentary level of physical activity during near and intermediate viewing. Several studies have suggested that bright outdoor light exposure is protective for myopia developmentet al.43, in 1994 as part of the ORINDA study, and has been utilized in several studies, albeit using different calculations26. For example, Zadnik, et al., determined diopter hours by adding together “three times the number of hours spent reading (for pleasure or studying), two times the number of hours spent playing video-type games, and the number of hours spent watching television,” and reported mean 53.8 ± 26.8 dh in eighth grade children14. Saw, et al., calculated diopter hours by multiplying various near work activities by the reciprocal of the distance for each activity, and reported ranges from 0.008 to 0.2126. Here, subjective diopter hours from the questionnaire averaged 25.67 ± 9.2 dh and objective hours from the RangeLife averaged 14.0 ± 4.0 dh. It is not unexpected that there would be discrepancies between subjective and objective diopter hours because of the variability in subjective data, and the differences in how subjective and objective diopter hours were calculated here. Therefore, the metric is best used as a relative measure to compare refractive error groups or across time periods when using a similar methodology.In this study, subjective and objective diopter hours were calculated to determine a near work index using each method. Diopter hours weights various near activities and viewing distances to determine a comprehensive value of near work exposure. The diopter hours metric was introduced by Zadnik, 38 and time outdoors37 have reported discrepancies between subjective reports and objective measures. We used the newly developed UH NEAR survey for subjective reports of visual activity. However, we still found variability between subjective and objective quantification of viewing distances, with most subjects overestimating time spent in near work as compared to objective measures derived from the RangeLife. These findings further justify implementation of objective measures to quantify environmental and behavioral factors. While objective measures are ideal for precisely quantifying visual activity, questionnaires will still be a beneficial supplement to objective measures to fully characterize behaviors.Studies employing objective wearable sensors to assess reading distance44, distance of reading material38, and other reading habits45.We found that the RangeLife device was capable of logging continuous measures of viewing distance with high sample rate (1 Hz) and no noise in the data. Continuous measurements with a high sample rate will allow for a more precise quantification of viewing distance, which, in future studies, can be analyzed in terms of the duration of continued near work with quantification of near viewing breaks that may be interspersed within the near work task. The absence of noise in the data eliminates the need for filtering and potential loss of data. Another important feature of range sensing devices is that they have a narrow beam diameter and can detect when the line of sight is directed at small targets, such as hand-held electronic devices. The beam diameter of the RangeLife is narrow enough such that it was able to detect hand held devices, books, and computer monitors. These characteristics are ideal for assessing near viewing behaviors that have been linked to refractive error, including the duration and frequency of near work38. Additionally, the authors found that the region of maximal sensitivity was about 30° for working distances of 40 to 100 cm. Therefore, small rotations from the line of sight are not expected to have a large impact on the measured viewing distance; however, future studies are aimed at quantifying the directional sensitivity for the RangeLife. Another limitation was that the device transmits data via a cord leading to an enclosure with a belt clip, which might present some difficulty with subjects who are involved in sports or other vigorous physical activity. Our adult subjects did not report problems in the current study, even during exercise. However, future instrument optimization is aimed at designing the components such that data can be stored within the sensor, eliminating the need for a wired enclosure.The RangeLife device presented some limitations in measuring viewing distance. The reliable measurable range was limited to 0.1 to 1.0 m, and therefore, distances greater than 1.0 m could not be quantified and were classified as “far” for the purpose of this study. Another limitation was that the device was mounted to a spectacle frame and therefore not necessarily aligned with the line of sight, which may have resulted in some inaccuracies as subjects rotated their eyes. We attempted to minimize this confounding factor by encouraging subjects to turn their head towards targets rather than rotating their eyes. A previous study using a similar device tested the directional sensitivity and found that the device retained its sensitivity even when the target was at a substantial angle from the measuring beamIn conclusion, the RangeLife was shown to provide continuous, objective, and reliable measures of near and intermediate viewing distances. Additionally, significant differences were demonstrated between RangeLife measures and traditional questionnaire data. The RangeLife device can be further implemented to assess viewing behaviors that may influence refractive error development in children and young adults.Visual Activity Survey


Magnesium is an essential mineral naturally present in the human body, where it acts as cofactor in several enzymatic reactions. Magnesium is a key cardiovascular regulator, which maintains electrical, metabolic, and vascular homeostasis. Moreover, magnesium participates in inflammation and oxidative processes. In fact, magnesium deficiency is involved in the pathophysiology of arterial hypertension, diabetes mellitus, dyslipidemia, metabolic syndrome, endothelial dysfunction, coronary artery disease, cardiac arrhythmias, and sudden cardiac death. In consideration of the great public-health impact of cardiovascular disease, the recognition of the negative effects of magnesium deficiency suggests the possible role of hypomagnesaemia as cardiovascular risk factor and the use of serum magnesium level for the screening and prevention of cardiovascular risk factors and cardiovascular diseases. Moreover, it might help with the identification of new therapeutical strategies for the management of cardiovascular disease through magnesium supplementation. In an adult human body, there are approximately 0.4 g Mg2+/kg [2+ represents a little percentage, less than 1% of all Mg2+ in the body [2+ is largely exchangeable to counteract acute changes in serum levels of this mineral, while one-third of skeletal Mg2+ accomplishes the same function [2+ from different types of food as green leafy vegetables, legumes, cereal and nuts, which have a great Mg2+ content, or fruits, meat, fish, and chocolate, providing a moderate amount of Mg2+. Moreover, water represents an important source of Mg2+, because it contains up to 30 mg/L of Mg2+ [Magnesium  [2+ intake and circulating Mg2+ with CV health [2+ intake in a large proportion of population, probably due to Western dietary habits. Additionally, other known causes of hypomagnesaemia, such as intestinal malabsorption, gastrointestinal losses, and diuretics or lassative assumption, are very frequent in general population but often underestimated and undertreated, particularly among elderly individuals.Scientific literature has reported the role of Mgs (CVDs) . More reV health –9; hypom2+ level suitable for the screening and prevention of CVD and opens new therapeutic scenarios with the possibility of reducing CV risk profile and treating CVDs through Mg2+ supplementation.The recognition of the possible role of hypomagnesaemia as risk factor for CV health, along with the underestimation of the importance of this mineral in daily clinical practice, makes serum Mg2+ exerts beneficial effects on the cardiovascular system by acting on transmembrane ion transport pumps, improving glucose and insulin metabolism, enhancing endothelium-dependent vasodilation, improving lipid profile, and acting as an antihypertensive and anti-inflammatory agent [2+ is a natural calcium antagonist, is an essential cofactor in mitochondrial oxidative reactions, and has anticoagulant and antiplatelet properties.Mgry agent . Additio2+ participates to the control of the activity of some ionic channels, such as sodium (Na+), potassium (K+), and calcium (Ca2+) [2+ reduces the rapid inward component of the delayed rectifier K+ channel (IKr) [2+ infusion provokes the slowing of atrioventricular (AV) nodal conduction and also determines the prolongation of PR and QRS duration [2+ prolongs both atrial and ventricular refractory period, reducing proarrhythmic substrate for triggered automaticity and reentry circuits [2+ deficiency is correlated with a prolonged QT interval potentially associated to the development of ventricular arrhythmias as the torsade de pointes. Magnesium sulfate (MgSO4) is usually successfully used during the episodes of torsade de pointes, because it is able to stop early after depolarizations (EADs) and automaticity by decreasing IKr current and blocking long-lasting type (L-type) Ca2+ activity [2+ channels, transient-type (T-type) and L-type [2+ exerts a protective effect against triggered activity, prevents coronary artery spasm, and plays a crucial role in modulating vascular muscle tone and, therefore, systemic arterial blood pressure. Furthermore, Mg2+ plays a critical role in the potassium-proton exchange and in the protection against potassium loss. Hypomagnesaemia weakens this mechanism and also promotes the augmentation of intracellular Na+ and Ca2+ concentrations. These concepts can explain how hypomagnesaemia can impact the physiological activity of muscle cells in general and cardiomyocytes and vascular muscle cells in specific, leading to cardiovascular diseases and coronary vasospasm [2+ overload that has detrimental effect on myocardial function. Competing for the same binding sites, Mg2+ may reduce intracellular Ca2+ overload during myocardial ischemia, and may restrict infarct size limiting coronary artery spasm, reducing postinfarction oxidative damage [Mgm (Ca2+) , 12. Mg2el (IKr)  and exerduration . Mg2+ prcircuits , 16. On activity . Throughd L-type , Mg2+ exasospasm . Additioe damage , and impe damage .2+ acts as a cofactor in multiple cellular reactions, some of which take place in mitochondria, that also represent the main intracellular reservoir of this ion and, through the Mg2+ transporter, mediate the redistribution of Mg2+ inside cells according to physiological stimuli [2+ has effects both on mitochondrial energy production and mitochondrial morphology, disrupting ATP production [Mg stimuli . Dysreguoduction .2+ seems to have beneficial effects on CV health, reducing the risk of developing type 2 diabetes mellitus and metabolic syndrome [2+ mediates pancreatic insulin release, postreceptor insulin signaling, and acts as a second messenger for insulin-mediated signal transduction [Mgsyndrome , 24. Thisyndrome  and enhasduction , 27.2+ has been associated to the regulation of inflammation and oxidative processes. Indeed, this ion has anti-inflammatory effect, modulating the release of nuclear factor-kB [2+ in the cardiovascular health.Mgactor-kB , and antactor-kB –34. The actor-kB , with th2+ has anticoagulant [2+ capacity to reduce platelet activation, both by inhibiting the production of platelet stimulating factors, such as thromboxane A2 (TXA2), and e increasing the release of platelet inhibiting factors, such as prostacyclin (PGI2) [2+ ions for specific sites in the glycoprotein (Gp) IIb subunit, modifying the receptor conformation and hindering fibrinogen-Gp IIb-IIIa interaction. This effect could be observed greatly in case of Mg2+ supplementation, as highlighted by Gawaz et al. [2+ is able to inhibit thrombin-induced Ca2+ influx [Mgoagulant  and antin (PGI2) , 38, andz et al. . Moreove+ influx .+ channel (K-ATP), as a possible substrate where endothelial dysfunction establishes itself. Starting from these considerations, it is more intelligible how Mg2+, as essential intracellular cation, can affect these mechanisms [2+ prevents vasospasm and endothelial damage [2+ levels affect and reduce the synthesis of vasodilator molecules and slow the proliferation of endothelial cells and promote the adhesion of monocytes, increasing the susceptibility of the CV system to oxidative stress and contributing to the creation of a prothrombotic and atherogenic surface [2+, infused after cardiac operations, affects endothelial-dependent vasodilation without interfere with NO production, and low extracellular Mg2+ levels affect NO-dependent vasodilation [2+ handling system, Mg2+ deficiency modulates vascular tone and can increase the risk of developing hypertension [2+ induces endothelial dysfunction reducing endothelial proliferation [Of note, hypomagnesaemia could increase the susceptibility to CVD affecting the endothelial function and modulating microvascular functions. The role of coronary microcirculation and its endothelial or nonendothelial-dependent dysfunction in the pathogenesis of CVD, and specifically of myocardial ischemia, is well known. Recent evidences in this field assigned a new visibility and importance to the presence of single-nucleotide polymorphisms (SNPs) in genes encoding for coronary ion channels, most of all for the ATP-dependent Kchanisms –43. Indel damage  by stimu surface , 44–59. dilation . By redurtension , 62. It feration . Particuferation , and, onferation , 65. Furferation . The uprferation .2+ is a fundamental mineral in cardiac health, and its role is essential in various different cardiovascular and metabolic conditions. It is a defense against the oxidative damage [2+ antagonist [2+ level, Mg2+ supplementation, and CVD. These evidences suggest that this ion could be monitored for screening and prevention of CVD and possibly supplemented as an adjunctive pharmacotherapy for CVD , T2D, and MetS. A recent meta-analysis demonstrated a linear dose-response relationship between Mgk of T2D ; Sarrafz of MetS . Throughlesterol , 84.2+ deficiency with CV risk factors promoting the generation of atherosclerotic plaques is probably the reason of the increased risk of ischemic heart disease (IHD) seen in patients with hypomagnesaemia. Particularly, the risk of myocardial infarction is also referable to the role that Mg2+ plays in favoring oxidative stress, suggested by a lot of evidences both in humans and animal models [2+ deficiency the lowering of plasma anticoagulants and an increased lipid peroxidation [2+ deficiency may promote oxidative injury in cardiovascular disease states and highlighted how actually low Mg2+ concentrations can accelerate and potentiate oxidative insult in the myocardium [2+ levels are associated with lower risk of IHD and fatal IHD [The association of Mgl models . Hans etxidation , while Kxidation . Kharb aocardium . The linatal IHD .2+ levels can bring to IHD is coronary vasospasm, and this event is mostly due to the consequences of hypomagnesaemia on intracellular Ca2+. A lot of studies have already demonstrated that Mg2+ deficiency is correlated to coronary artery spasm [2+ status in and out of cells in 12 women affected by variant angina, found that Mg2+ concentration in the cytoplasm of erythrocytes was well related to the activity of the variant angina [2+ could be useful in the prevention of coronary vasospasm in patients with vasospastic angina. They demonstrated that Mg2+ infusion can induce nonsite specific basal coronary vasodilation and block coronary spasm acetylcholine-induced in patients affected by vasospastic angina [Another mechanism through which low Mgry spasm –90. Guo t angina . Startinc angina .2+ on arterial stiffness, a known marker of CV health. They found that Mg supplementation reduced the arterial stiffness without any effects on blood pressure values, suggesting a potential way through which an increased Mg2+ intake could exert benefits for cardiovascular health [Recently, Joris et al. conducted a randomized, double-blind, and placebo-controlled intervention trial in a group of overweight and obese adults to study the effects of Mgr health , 92.2+ and risk of cardiovascular disease, such as ischemic heart disease, and it was confirmed with significant results, supporting recommendations to increase Mg2+ intake, mostly the consumption of Mg2+-rich food, as vegetables and nuts [A systematic review and meta-analysis conducted by Del Gobbo et al. tried to draw a link between circulating and dietary Mgand nuts .2+ supplementation could have beneficial effects in the early phase of acute coronary syndrome and after implantation of drug-eluting stent (DES). Gyamlani et al. found that Mg2+ levels during and after acute myocardium infarction (AMI) were lower in patients affected by AMI than controls and that intravenous Mg2+ administered in the immediate postinfarction period in the patients had a cardioprotective effect and the capacity to reduce arrhythmias, pump dysfunction, and death [2+ levels could be considered, independently from other risk factors, as predictor of major adverse cardiac events (MACEs) in patients who undergo a DES implantation after AMI, but not unstable angina [2+ as independent predictor of electrocardiographic no-reflow and long-term mortality in 111 patients after ST-segment elevation myocardial infarction (STEMI) and primary percutaneous coronary implantation (p-PCI) [2+ levels and acute stent thrombosis after a p-PCI in people with STEMI. The results pointed out that Mg2+ was an independent predictor of acute stent thrombosis in these groups of patients [2+ represents an important ion in the determination of membrane potential, dosing its plasma levels, together with calculation of the concentrations of the other main ions, could be useful in the identification of the membrane permittivity and conductivity, for the purpose of better understanding the mechanisms that are at the basis of the hemocompatibility of vascular stent materials [New evidences suggest that Mgnd death . A studye angina . Similar (p-PCI) . In contpatients . Besidesaterials .2+ in the reduction of mortality after acute myocardial infarction are not all concordant, and there are conflicting results, such as the MAGIC (Mg2+ in coronaries) trial that did not report any advantage of early Mg2+ administration in addition to the standard therapy in high-risk patients after STEMI, with no effects on 30-day mortality [2+ levels in predicting hard events  in patients after AMI [2+ supplementation could exert the beneficial effect in low-risk patients with CVD or in prevention of CVD rather than in patients with high CV risk profiles.Nevertheless, data regarding the protective role of Mgortality . Vassallfter AMI . The res2+ content or the lower Mg2+ concentrations in people who die of sudden death. A possible explanation of this correlation is referable to cardiac arrhythmias and coronary vasospasm, which are usually associated to Mg2+ deficiency. An important study related to this question was conducted by Peacock et al. in a cohort of more than fourteen thousand persons, distributed according to the value of their serum Mg2+. The results showed that people at the higher quartile of serum Mg2+ had a significantly lower risk of sudden death compared to individuals at the lower quartile, and these findings were confirmed also after the adjustment for the major potential confounders of Mg2+-SCD relationship, like hypertension, diabetes, serum K+ concentrations, or use of diuretics [Finally, a big number of studies published in the past decades links hypomagnesaemia to sudden cardiac death (SCD) because of different reasons, such as the major frequency of sudden death in areas where community water supplies have a little Mgiuretics .2+ level for screening and prevention of CVD and Mg2+ supplementation as adjunctive pharmacotherapy for patients suffering from heart disease.All these evidences suggest that hypomagnesaemia may predispose to the development of CV risk factors and CVD. The awareness of the possible role of this ion as risk factor for CVD may lead to use Mg2+ does not unequivocally reflect the intracellular concentration of this ion, which better correlates with Mg2+ functions [2+ within lymphocytes and erythrocytes would be likely more accurate and would correlate better with intramyocardial Mg2+ [2+ intake; thus, it is not possible to discriminate among the effect of Mg2+ and residual effect derived from the intake of other microelements. Moreover, it is not possible to determine the amount of supplemented Mg2+ effectively absorbed and utilized. Another limitation is the different types of Mg2+ formulations used for the supplementation. The studies about the effect of Mg2+ intake on CV health use different Mg2+ formulations, both organic and inorganic, but it have been demonstrated that there are differences in the bioavailability among formulations. These differences could influence the results of the studies. Lastly, some cited meta-analyses present heterogeneity between the studies, possibly affecting the reliability of statistical analysis. These limitations suggest that new large prospective randomized trial is necessary to elucidate the association among Mg and CV health and to assess the benefits and the usefulness of Mg supplementation in the prevention and treatment of CVD (Figures Nevertheless, the aforementioned studies have several limitations. First of all, serum Mgunctions , 102, thial Mg2+ . Secondl Figures .2+ and cardiovascular health has been clearly demonstrated and underlines the possible pathophysiological mechanisms through which Mg2+ deficiency can promote the onset, progression, and maintenance of CVD. In fact, hypomagnesaemia influences negatively the CV health and is associated with an augmented incidence of hypertension, type 2 diabetes, dyslipidemia, atherosclerosis, arrhythmias, and coronary artery disease. In consideration of the great public-health impact of CVD, the recognition of the detrimental effects of Mg2+ deficiency on CV health suggests the possible role of Mg2+ blood levels for the screening and prevention of CV risk factors and CVD and helps the identification of new therapeutical strategies for the management of CVD through Mg2+ supplementation. This concept is even more shareable if framed in a wider and more complete perspective, that aims to embrace in a global way any possible alteration or disease, even noncardiovascular ones, which can damage the heart health and whose treatment or correction can support specific cardiological therapies, from the standard one to the most innovative, in the pursuit of the healing and the well-being of the patients [2+ level assessment in cardiovascular patients and in the general population and the usefulness of Mg2+ supplementation in the prevention and treatment of CVD.The present review shows that a relationship between Mgpatients –106. Con