The ability to know where our body parts are located in space (proprioception) is fundamental for both successfully interacting with the external world and monitoring potential threats. In this case-control study, we investigated whether the absence of proprioceptive signals may affect physiological defensive responses. To this aim, a right brain-damaged patient with a left upper-limb proprioceptive deficit (P+ patient) and age-matched healthy controls, underwent the recording of the Hand-Blink Reflex (HBR). This defensive response, elicited by electrical stimulation of the median nerve and recorded from the orbicularis oculi, is modulated by the hand position: it is enhanced when the threatened hand is near to the face, inside the defensive peripersonal-space (DPPS). According to the classical neuropsychological perspective, we used P+ patient as a model to investigate the role of proprioception in HBR modulation, by manipulating the congruity/incongruity between the intended and actual positions of the stimulated hand. P+ patient, with his eyes closed, had to voluntarily place his left hand either far from or near to his face and to relieve the arm’s weight over a supporting device. Then, in congruent conditions, the hand was stimulated in the actual (intended) position. In incongruent conditions, the patient’s hand was moved by the examiner from the intended to the opposite (not-intended) position and then stimulated. We observed an inverse response pattern between congruent and incongruent conditions. In congruent conditions, P+ patient showed an HBR enhancement in near compared to far position, comparable to that found in healthy controls. This suggests that, even in absence of proprioceptive and visual information, the HBR modulation was still present. Conversely, in incongruent conditions, P+ patient showed a greater HBR magnitude for far position  than for near position . This result suggests that proprioceptive signals are not necessary for HBR modulation to occur. It relies more on the intended than on the actual position of the hand. The role of motor intention and planning in shaping the DPPS is discussed. The development of a coherent representation of one’s own body in space requires the combination of motor commands , somatosSince external stimuli signaling potential threats may suddenly approach the body, a continuous assessment of environmental threats according to a body reference frame, is essential to survive. The closer the threats occur, the more likely they are to cause damage. Consequently, defensive and avoidance reactions are stronger. Thus, the boundary of the DPPS can be described by looking at the amplitude of defensive responses to aversive stimuli, occurring at different locations with respect to the body. In monkeys, the electrical stimulation of the ventral intraparietal area (VIP) and a polysensory zone (PZ) in the precentral gyrus, evokes defensive responses. It is as if the monkeys are defending the body part where the sensory receptive field of the neuron is located . This, aIt has been demonstrated that, in order to modulate the HBR, online visual information is not necessary. Indeed, when the hand is stimulated near to the face, the response enhancement occurs even in healthy subjects with their eyes closed  are both available to estimate the final position of the hand . Thus, iad hoc paradigm devised to investigate the role of proprioception in the HBR modulation, by manipulating the congruity/incongruity between the intended and the actual position of the stimulated hand . Free MRIcron software , the anterior limb of the internal capsule, the anterior corona radiata and the external capsule , who were all right-handed , were en∗2 factorial design, with four experimental conditions, namely “far-congruent” and “near-congruent,” in which the intended position and the actual positions of the hand were the same; “far-incongruent” and “near-incongruent,” in which the intended position and the actual position of the hand were the opposite. The study consisted of four blocks where experimental conditions were presented in a counterbalanced order . Each block lasted for 3 min and consisted of a total of 6 trials; 3 in the far position and 3 in the near position in alternating trials, resulting in a total of 24 trials (12 far and 12 near) per condition. It is important to note that, for the purpose of the study , visual information was precluded. Thus according to a previous study  or near to the face . In each trial, the experimenter asked the patient to reach one of the two positions  and the patient voluntarily performed the postural manipulation. Then, he had to keep the arm relaxed, unloading the arm’s weight over a dedicated device able to sustain it, and to wait for the electrical stimulus delivered to the median nerve (see details in Stimulation and Recordings). The device was specifically built to fix the patient’s forearm over a soft panel and sustain the elbow by means of an adjustable extension able to completely support the arm’s weight. The device was connected to a pulley, which allowed the co-experimenter to move the patient’s arm in space. The postural manipulation was performed in two different conditions, depending on the congruity/incongruity between the intended and the actual positions of the hand. According to the condition, the supporting device was kept in the same position requested to the patient (congruent condition) or moved by the co-experimenter in the opposite one (incongruent condition); see Figure Healthy participants underwent the classic HBR paradigm, in which the stimulated hand was voluntarily placed either far from or near to the face ,b. In thElectromyographic activity (EMG) was recorded from the orbicularis oculi muscle bilaterally, using two pairs of bipolar surface electrodes. The active electrode was placed over the mid lower eyelid and the reference electrode laterally to the outer canthus. EMG signals were amplified and digitized at 10 kHz , and stored for offline analysis. The HBR was elicited by delivering transcutaneous electrical stimuli to the left median nerve, using a surface bipolar electrode . In each participant, by increasing the stimulus intensity until a clear and stable HBR was observed in at least five consecutive trials or the participant refused a further increase in intensity, we calibrated the stimulus intensity able to elicit a clear blink reflex . The stimulus duration was 200 μs and to minimize stimulus habituation the inter-stimulus interval was ∼30 s, following prior methods .1 and Letswave2 software . Then, an index of the HBR modulation, calculated as the difference between hand positions (Δ near-far), was used as a dependent variable to perform case-control comparisons by means of the Crawford’s test  = 6.28; p = 0.0001; power = 0.99; HBR magnitude: mean ± SD; far: 11.97 ± 8.93; near: 14.57 ± 10.06].In the healthy subjects group, as expected, the paired t = 0.36; p = 0.36; Z-CC = 0.38; 95% confidence interval = -0.215 to 0.959; mean ± SD Δ near-far congruent controls = 2.59 ± 1.58; Δ near-far congruent P+ patient = 3.19).In the case-control comparisons, the Crawford’s test revealed no significant differences between the P+ patient’s congruent conditions and controls; i.e., the P+ patient showed an HBR enhancement in the near compared to the far condition, comparable to that found in the healthy controls ; see Figure On the contrary, the Crawford’s test revealed significant differences between the P+ patient’s incongruent conditions and controls; i.e., the P+ patient showed an opposite response pattern in comparison to the controls, with greater HBR values for the far position  than for the near position   versus sensorimotor feedback (afferent information) in shaping the HBR magnitude. This is a rare case of a patient with spared tactile and nociceptive sensibility  and spared motor ability , but with a selective deficit of proprioception. Indeed, even if he could perceive the electrical stimuli delivered to the median nerve and he was able to verbally report that the electrical stimulation occurred on his left wrist, he was not able to use this somatosensory information to localize the position of the affected hand. Thus, according to the classical dichotomy between body image  and body schema  , we can From an anatomical point of view, the P+ patient’s brain lesion is compatible with the findings of previous literature about the proprioceptive loss in stroke patients and the neural correlation of position sense in healthy participants. With respect to the basal ganglia, they are traditionally related to a series of motor functions, including an important role in intentional motor programming, known to exert a modulatory effect on the motor cortex activity . CertainThis pathological condition, with the spared motor component and affected position sense, allowed us to describe an important new finding. As proved by the P+ patient’s results in the congruent conditions, even though proprioceptive information was lost (due to the brain lesion) and visual information was precluded (due to the patient having his eyes closed), the HBR modulation was still present. This suggests that even if proprioceptive information can be sufficient for HBR modulation to occur (see above), it is not necessary. In the P+ patient, the HBR modulation occurred even when the ability to sense the limb position was completely lost.In the framework of proprioceptive deafferentation, other than motor intentionality, embodiment processes also assure eliciting of the HBR. An our previous study, which investigates HBR in a pathological context, suggested that HBR modulation is present in brain-damaged patients with lost position sense . HoweverIn the present study, the visual feedback was precluded , so that the HBR modulation we observed in the P+ patient could only be driven by the patient’s beliefs about the hand position, depending on the voluntary motor program implemented to reach the requested position (either up to or down from the face). This was clearly evident in the incongruent conditions, when the HBR was greater in the far position  than in the near position . Therefore, in the P+ patient, it seems that the HBR modulation relies more on the intended than on the actual position of the hand.From an evolutionary perspective, it makes sense that a defensive response, such as the HBR, can be modulated by several different factors. Within these factors, together with primary sources of information such as vision and proprioception, motor intention and planning (informing us about the predicted position of the hand) can play a crucial role.CF and FG designed the research and analyzed the data. CF, VB, and FG performed the research and prepared the figures. CF and VB performed patients’ lesion mapping. PG recruited patients and performed neuropsychological assessment. All authors wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This study reviews qualitative research into the sociocultural meanings and subjective experiences that midlife men in the United Kingdom (UK) associate with their drinking. In the UK, average weekly alcohol consumption is highest among midlife men, and they are disproportionately affected by alcohol harm. There is increasing recognition that public health messages to support behaviour change must be based on an in-depth understanding of drinking motivations and experiences.Systematic literature review of studies exploring motivations for and experiences of drinking among UK men aged 45–60 using qualitative methodology. Medline, PsycINFO and the Social Science Citation Index were used, along with manual searches of key journals, Google searches and a call for evidence. The Critical Appraisal Skills Programme tool was used to quality-assess papers. Thematic synthesis was used to combine and analyse the data.From 5172 titles and abstracts (1995–2018), 11 publications were included, representing 6 unique studies. Five themes were identified: ‘Drinking Motivations’; ‘Drinking Justifications’; ‘Drinking Strategies and Control’; ‘Social Norms and Identity’ and ‘Harm’. Motivations for drinking among midlife men were associated with relaxation, socialising and maintenance of male friendships. They justified drinking as a choice and emphasised their ability to meet responsibilities, which they contrasted with ‘problem drinkers’. Social norms governed drinking behaviours as an expression of masculinity.This review highlights the significance of the meanings and social importance of alcohol consumption among midlife men. Interventions using information and guidance should consider these when aiming to effectively influence the way this group drinks. •Motivations for drinking among midlife men were associated with relaxation, socialising and maintenance of male friendships.•Midlife men saw their drinking as a choice and in contrast to ‘problem drinkers’ who cannot meet key responsibilities.•Strong social norms govern drinking amongst this group, with alcohol consumption seen as an expression of masculinity.•There is an evidence gap of primary studies focussing on drinking in middle-aged men. CampaigA survey of a UK representative sample of adults sought to capture such insights into drinking attitudes and behaviours . Based oThis paper sets out to systematically review existing qualitative research into the sociocultural meanings and subjective experiences that midlife men in the UK associate with their drinking, in order to inform efforts to encourage these men to reduce their drinking to less harmful levels.22.1Addiction, Alcohol and Alcoholism, and Addictive Behaviours. A call for evidence was sent to experts in the field and issued on the Drinkaware  website. For all included studies, a forward citation search was conducted using ISI Proceedings (Web of Science). The reference lists of all included publications were screened.Database searches were conducted in Medline, PsycINFO and the Social Science Citation Index from January 1995 to April 2018, restricted to those published in English. Search terms included “alcohol*”, “drunk*”, “motivat*”, “behav*”, and “attitud*” as well as MeSH terms where appropriate. Terms were combined using “AND” or “OR”. See 2.2Study selection involved two phases. Based on title and abstract papers were excluded if they did not explore motivations for, or experiences with, alcohol consumption; exploration of drinking motivations or experiences was required to be either an aim of the study, or a substantial finding in the results. To ensure an in-depth understanding and rich description of motivations and experiences only studies presenting primary data using qualitative methods were included. Participants were required to include midlife men (aged 45 to 60) living in the UK, to ensure their experiences and views were meaningfully captured as relevant to the specific life course and sociocultural context . Studies were excluded if published in a non-English language, prior to 1995, or including a sample of exclusively lifetime abstainers, or people with a past or present alcohol addiction. These exclusion criteria were introduced in order to ensure that experiences and views were current (as attitudes and behaviours may change over time) and relevant to the defined population . After full text screening papers were excluded if findings could not be identified as relating either specifically to midlife men or be relevant across age and gender groups.Where different publications from the same research were identified , either the most recent or the version with the focus most closely aligned to the aims of this review was included.An initial screening was undertaken of papers published January 1995 to July 2015. 10% of titles and abstracts were screened by two reviewers (HP and MM), with an inter-rater agreement of 98%. Consensus was established through discussions with co-authors (JLa and AR). The remaining screening was conducted by one reviewer (HP or MM). A second screening was undertaken of papers published August 2015 to April 2018, and again 10% of titles and abstracts were screened by two reviewers (ACM and JLa), with an inter-rater agreement of 97%, and the remaining screening conducted by one reviewer (ACM). All full-text papers were screened independently by two reviewers . Inter-rater agreement was 88%; in the 10 instances of disagreement a third reviewer (JLa or AR) acted as arbitrator.2.3The quality of included studies was assessed using the Critical Appraisal Skills Programme (CASP) criteria for qualitative studies . For eacData were extracted from the papers using a purpose-built extraction table. Data extraction was performed by one review author (MM) and checked by a second (HP). The findings of each study was taken to be all text under the heading ‘findings’ or ‘results’, including, but not limited to, illustrative quotes . See S3 2.4Thematic synthesis was used to combine and analyse the data . The parThe first step of the process of thematic synthesis was line by line coding of the data. Free codes were applied, with in vivo codes used wherever possible. The reviewer (MM) adopted an iterative approach, continually adding to the bank of codes, revisiting old codes, and merging or revising existing codes where necessary. A second reviewer (HP) coded a sample of the data (n = 9), providing a second check and constructively challenging and questioning codes . The fin3The database searches produced a total of 5172 results after duplicates were removed. 5091 were excluded after title and abstract screening and additional three unique papers were identified for inclusion via hand searches, expert call for evidence, Google Scholar, forward citation searching and reference list scanning, leaving 84 papers to be screened in full. After full text screening 73 papers were found not to meet the inclusion criteria, leaving 11 publications included in the review. See 3.1The 11 publications were published between 2002 and 2014, and represent six unique studies. The studies took place across England and Scotland, with two unique studies conducted in the North East of England : one uniOnly one publication included a study population of exclusively men in middle age (defined by study authors as 30 to 50) , two othOne unique study stated that participants were socioeconomically diverse , and anoThree unique studies provided information on the alcohol consumption of participants. Using the UK definitions in place at the time, one study included drinkers classified as low risk  or 21  units/week, with one UK alcohol unit defined as 8 g pure alcohol), hazardous (>14 or >21 units/week), or harmful (>35 or >50 units/week) . In anotEthnicity was addressed in two unique studies: one only included White participants  and anotSix publications used focus group methodology ; three eThe quality of the publications based on the CASP criteria ranged from 18  to 13 F out of a3.2Five themes were identified: ‘Drinking Motivations’; ‘Drinking Justifications’; ‘Drinking Strategies and Control’; ‘Social Norms and Identity’ and ‘Harm’. These will now each be discussed in turn. See 3.3This theme explores the reasons participants gave for drinking. The sub-themes identified were: drinking to relax and its distinction from drinking to cope; drinking to socialise and its importance in establishing and maintaining friendships; and drinking to get drunk.Drinking was widely described as a means of relaxing and unwinding . For somFor many men, drinking was the central focus on which many friendships and relationships were established and maintained. Drinking was widely regarded as a highly sociable activity associated with having fun and bonding with others. Examples of the perceived positive, sharing nature of drinking included splitting a bottle of wine over dinner at home , buying Drinking with the intention of getting drunk was also identified in a number of studies . Some re3.4A common theme was the importance of justifying alcohol consumption. Three key sub-themes were: drinking as a controlled choice; meeting responsibilities; and ‘othering’ of problematic drinking as something unrelated to own identity.Drinking was presented as something done out of choice. Participants emphasised that they were able to control and restrain their drinking, and in this way asserted that their alcohol consumption was unproblematic . Some miCommonly, participants talked about an ‘other’ kind of drinker whom was a cause for concern, contrasting this ‘problematic other’ to their own, unproblematic drinking. Young drinkers were characterised as less experienced and with lower alcohol tolerance. Their drinking was associated with an explicit aim of getting drunk quickly, increased aggression and causing public nuisance. This youthful drinking was contrasted with the ‘civilised’ drinking of the middle aged . Partici3.5Participants described the strategies they used to reach an optimal state of intoxication, how it was achieved and the consequences associated with failure to achieve it. The following sub-themes were identified: drinking strategies; being ‘in the zone’; knowing when to stop; drinking more with age and feeling superior to younger men; passing the ‘point of no return’; and loss of control.Some participants used particular drinking strategies to enhance their drinking experience and reduce negative consequences of excessive drinking. These included eating certain foods to moderate the effects of alcohol and reduce the likelihood of making oneself vomit so as to ‘keep drinking’ . In addiThe second sub-theme of being ‘in the zone’ relates to a pre-planned, optimal, intoxicated state which participants associated with enjoyment and used to justify their drunkenness. The participants expressed their desire to reach this state and revealed that it was difficult to achieve it, as various factors, such as social context and surroundings could influence this process .Knowing when to stop drinking is based on experience and monitoring physical changes, rather than on factors such as counting units. Some men described this knowledge as being ‘subconscious’, and attributed it to being an ‘experienced’ drinker able to control their alcohol consumption . Others Midlife men expressed feelings of superiority, viewing themselves as being more experienced drinkers, able to drink more and be in control of their behaviour, as opposed to younger men who might find it hard to ‘keep their cool’ . This liHowever, having reached an optimal state of intoxication, some participants described that they continued to drink and attributed it to loss of control, as they felt that they could not stop despite experiencing various negative effects. The participants used metaphors to describe the powerful effect of alcohol on their behaviour as they ‘went with the flow’ and ‘got bladdered’ without wanting to do so . Further3.6Drinking behaviours were guided by strong social norms existing within social groups and drinking alcohol linked to specific identities. There are two key sub-themes to consider: alcohol as a symbol of masculinity; and social judgements on price and quality of alcohol.Strong associations were identified between masculinity and specific aspects of the purchasing and consumption of alcohol. Drinking pints of beer in the pub with friends was widely recognised to be a masculine activity – one which was highly valued . DrinkinThe selection of a certain type of drink could influence an individual's sense of identity and self-esteem. Wine was described as more ‘classy’ or ‘sophisticated’ by some , and exp3.7The studies illuminated perceived and experienced harm associated with alcohol consumption in midlife, beliefs surrounding the drinking guidelines and views about moderation and reduction of consumption. The following sub-themes were identified: negative psychological and physical effects of drinking; concerns surrounding male mental health and emotional vulnerability; link between alcohol and aggression; interpretation of guidelines; rejection of harmful effects on health; reasons for reducing consumption and perceived drawbacks and benefits of drinking.Participants had experienced various negative psychological effects of excessive drinking, such as exacerbation of negative mood states, humiliation , uncontrSome men described ‘session depression’ and ‘the Sunday blues’, and exacerbated negative mood (e.g. guilt) following a heavy drinking session . In the The link between alcohol and aggression was also identified in some studies. Alcohol was identified as a causal factor (‘like a fuse…waiting to go off’ ) in releParticipants expressed cynicism and rejection of the notion of units  and guidThe reasons midlife drinkers described for reducing their alcohol consumption included health concerns, such as a recent hospitalisation and ageing ; realisaA final sub-theme captures a sense by participants that the drawbacks of drinking are outweighed by the benefits, which may to some extent explain the maintenance and normalisation of heavy drinking in midlife .4The qualitative studies reviewed in this paper highlight the significance of the meanings and social importance of alcohol consumption to midlife men in the UK, consistent with wider research on how social and cultural factors influence the specific pattern of alcohol consumption within a society . The revThe midlife men in the reviewed studies use elaborate strategies to manage their drinking to maximise the beneficial experiential and social effects; but while these for some involve clear boundaries regulating when to stop drinking to avoid negative experiential and social effects, for others (or at other times), the perception of their own ability to control their drinking is not as pronounced. A consolation for the midlife men is, however, that they generally feel that they now are much better able to control this compared to when they were younger. The men feel that they strongly benefit from their identity as ‘drinkers’ in terms of the cultural norms of masculinity and strength it is associated with, as found in other research . The malThe role of drinking, and drinking particular types of drinks, as affirmation of socially constructed notions of masculinity  resonateWhile the evidence reviewed suggests that UK midlife men have some knowledge of the harms that can be caused by drinking alcohol, it is mainly short-term harms (such as hangovers and effects on mood) they are concerned about, and they mainly associate longer-term health harms with the abovementioned ‘problem drinker’. In agreement with recent research on attitudes to alcohol guidelines , the rev4.1Only one of the included papers drew on data exclusively from midlife men, which could mean that insights pertaining specifically to these were diluted. Furthermore, the findings highlighted here relate to a group of middle-aged men from a diversity of geographical regions within the UK. Participants were also diverse with regards to their levels of alcohol consumption, spanning from low risk or ‘occasional’ drinkers, to those classified as harmful or dependent drinkers. Information regarding socioeconomic status and ethnicity were less well reported by primary study authors, but there is indication that some study authors did attempt to achieve diversity in these respects. The extensive reliance on focus groups is a limitation as this methodology, while good at exploring social norms, is less useful for revealing individual motivations and experiences. A limitation of the thematic synthesis approach is that it does not allow investigation of the richness of the original full qualitative data sets, but accesses the insights through the analytical perspectives and illustrative data selections applied by the authors of the original studies. However, this third level interpretation  identifi5The evidence of increasing harm experienced among midlife men in the UK as a result of excessive drinking provides a strong rationale for research exploring the experiences of and motivations for drinking in this group. The findings presented in this review make it clear that any intervention built around information and guidance aiming to effectively influence the way these men drink would have to carefully consider insights concerning the cultural meanings and social importance they associate with their alcohol consumption.Full Medline search strategy.Supporting information 1CASP quality appraisal tool for qualitative research (adapted).*Supporting information 2Data extraction fields.Supporting information 3Why the primary study is relevant to a review of drinking in middle-aged men.Supporting information 4Detailed CASP quality results.Supporting information 5Illustrative quotes.Supporting information 6The following are the supplementary data related to this article.No research ethics approval was required for this review study as it did not involve the collection of new empirical data. No dedicated research funding was received as the authors did the work as part of their day job research roles: three as employees of academic institutions , one as a student placement (MM), and three  as employees in the research function of the UK-wide alcohol education charity Drinkaware. The charity is established through an agreement between the UK government and the alcohol industry, and is funded primarily by voluntary and unrestricted donations from UK alcohol producers, retailers and supermarkets. Drinkaware is governed independently and works in partnership with others to reduce alcohol-related harm by helping people make better choices about their drinking.


Body-fluid-exposed bioactive glasses (BGs) integrate with living tissues due to the formation of a biomimetic surface layer of calcium hydroxy-carbonate apatite (HCA) with a close composition to bone mineral. Vast efforts have been spent to understand the mechanisms underlying in vitro apatite mineralization, as either formed by direct precipitation from supersaturated solutions, or from BG substrates in a simulated body fluid (SBF). Formally, these two scenarios are distinct and have hitherto been discussed as such. Herein, we contrast them and identify several shared features. We monitored the formation of amorphous calcium phosphate (ACP) and its crystallization into HCA from a Na After 2onset of apatite formation; a qualitative relationship between the apatite-formation degree and the P concentration of the solution is well-documented in the literature  ≲ [Ca] ≈ 10 mM. At a BG surface, on the other hand, CaP is nucleated heterogeneously at low supersaturation conditions in an SBF solution  bioactivity.This section highlights several apparent similarities between apatite formation by spontaneous precipitation from Ca/P-bearing solutions with HCA production at BG surfaces in SBF/buffered water. These two scenarios are = 1.0 mM ) that isidentical sigmoidal growth characteristics as those well-established for spontaneous H(C)A precipitation  values, as witnessed by Starting at the onset of H(C)A formation, the “proliferation” interval is responsible for the majority  ACP formation and ACP→HCA transformation processes from the SBF-soaked BG (see throughout the proliferation period (12 h < both ACP and H(C)A growth processes occur largely simultaneously [Notably, while d BG see : during aneously ,26. Yet,amount of apatite and mainly a progressively enhanced structural ordering of the apatite crystallites  [Apatite precipitation studies demonstrate that the maturation period is characterized by a rather modest increased ipening) , accompaipening) ,7. Theseminor ACP production during the former interval, but largely simultaneous ACP formation and ACP→HCA crystallization events throughout the proliferation, during which a majority of all CaP phases form. This feature revises the HM by partially merging its two last steps (4 and 5), as discussed further in Both the evolution of the P concentration in the solution d and the5 system ,48,67, a [2–P2O5  systems,[2O–SiO2 ,48, and –SiO2 [2  glasses;2 [2 [2) ,79, whicThe above-cited papers ,72,75,79both ACP and HCA phases grew during 24–72 h of SBF exposure .Ohtsuki et al.  soaked gntration . We estiapparent sole presence of ACP due to a markedly retarded—or even quenched—ACP→HCA conversion, as discussed further in refs. [We conclude that most literature reports of apatite formation from BG surfaces are indeed consistent with only a minor ACP production before the onset of HCA crystallization, followed by largely coincident ACP/HCA formation processes. Yet, there are a few exceptions ,80 that in refs. ,53,70,71formally only glasses that incorporate both Ca and phosphate species may produce HCA, the HM is also valid for in vitro testing in SBF media [(1)First, a rapid exchange occurs of the Ca(2)The silicate network at the surface fragments due to hydrolysis of Si–O–Si bonds according to Si–O–Si + H(3)2), implying that Si–O–Si bonds form via condensation of neighboring SiOH moieties  glass is exposed to aqueous solutions was proposed by Hench  mainly oBF media ,57 as weBF media , assuminhydrated silica gel surface layer rich in SiOH groups but depleted in Ca(4)“Migration of Ca(5)According to Hench, the subsequent ACP→HCA transformation proceeds as “crystallization of the amorphous CaO–PThe first three HM steps only involve reactions between silicate surface species and the surrounding solution. They altogether lead to a  cations ,41,72,81cleation ,41,72,81identifying the "amorphous CaP film/layer" with ACP, its close correspondence to the well-established apatite precursor observed from spontaneous precipitation follows directly. Despite that this interpretation is already recognized by some researchers in the BG community and has experimental support  A clear boundary between the two “silica gel” and “CaP” layers do not generally exist, where gradients of the Ca, P, and Si concentrations are usually observed outwards from the core of the glass [ Except for stipulating that the ACP nucleation is triggered by the “silica gel” layer [ Stage 5 of the HM suggests that the incorporation of “foreign” ions (such as Na(CP phase .iv) The large surfaces of sol-gel-derived BGs—and notably so MBGs with ordered arrangements of mesopores [:On the basis of new experimental data from an SBF-exposed Nai) HCA forms via an ACP precursor with very similar  chemical/structural nature as that preceding HCA formed from solutions supersaturated with respect to apatite. (ii) For increasing SBF-exposure intervals, the phenomenological apatite formation manifests the three-stage sigmoid-type growth of consecutive induction, proliferation, and maturation periods; however, solely the induction period is hitherto recognized and discussed in the literature on in vitro HCA formation from BGs because it reflects the “in vitro bioactivity” of the glass. (iii) The feature of “autocatalytic” apatite growth during the proliferation stage accounts for the majority of the apatite production, and for the SBF-exposed glass examined herein, implying ≈90% and ≈10% of the total HCA formation occurring during the respective proliferation and maturation stages. literature data on HCA formation in buffered water/SBF from BGs with widely differing compositions.simultaneous ACP/HCA formation processes, implying that a relatively modest (relative) amount of ACP forms during the induction interval, whereas the main portion emerges during the initial stages of the proliferation period, despite the rapid and concurrent ACP→HCA crystallization. This partially revises Hench’s mechanism by merging its last two events of as-proposed consecutive layer formations [identical phenomenological HCA growth proceeds. Consequently, once the first embryos of ACP are nucleated—either homogeneously or heterogeneously—these scenarios might also share the same mechanistic details of the ACP→HCA transformation. These lines are worth exploring further, as they may unify the homogenous and heterogenous nucleation concepts and potentially reveal a universal apatite-growth model, while some aspects of the biomimetic apatite formation at BG surfaces may be learned from the comparatively more studied case of directly precipitated apatite.We also discussed our findings in view of the scheme proposed by Hench  for in vrmations  of prima


Videographic material of animals can contain inapparent signals, such as color changes or motion that hold information about physiological functions, such as heart and respiration rate, pulse wave velocity, and vocalization. Eulerian video magnification allows the enhancement of such signals to enable their detection. The purpose of this study is to demonstrate how signals relevant to experimental physiology can be extracted from non-contact videographic material of animals.We applied Eulerian video magnification to detect physiological signals in a range of experimental models and in captive and free ranging wildlife. Neotenic Mexican axolotls were studied to demonstrate the extraction of heart rate signal of non-embryonic animals from dedicated videographic material. Heart rate could be acquired both in single and multiple animal setups of leucistic and normally colored animals under different physiological conditions  using a wide range of video qualities. Pulse wave velocity could also be measured in the low blood pressure system of the axolotl as well as in the high-pressure system of the human being. Heart rate extraction was also possible from videos of conscious, unconstrained zebrafish and from non-dedicated videographic material of sand lizard and giraffe. This technique also allowed for heart rate detection in embryonic chickens in ovo through the eggshell and in embryonic mice in utero and could be used as a gating signal to acquire two-phase volumetric micro-CT data of the beating embryonic chicken heart. Additionally, Eulerian video magnification was used to demonstrate how vocalization-induced vibrations can be detected in infrasound-producing Asian elephants.Eulerian video magnification provides a technique to extract inapparent temporal signals from videographic material of animals. This can be applied in experimental and comparative physiology where contact-based recordings  cannot be acquired. Fick’s convection equation for the cardiovascular system } is constant or varies in a predictable way with changes in fH, then measurements of fH can be used to evaluate MR of animals in both laboratory and field conditions , a Mexican caudate amphibian renowned for its tissue regenerative potential [fH ], and by selecting a specific channel  of the magnified video with standard 8-bit RGB colors. Additionally, we wanted to demonstrate a simple cardiovascular physiology relevant setup in which EVM-acquired fH could be obtained for resting, exercised, and anesthetized axolotls. We studied six leucistic axolotls  that were video recorded from a dorsal viewpoint after 48 h of fasting and 24 h of undisturbed rest  from the thoracic area resulted in escape behavior. Following an additional 24 h of undisturbed rest, benzocaine was added to the aquaria via catheters to induce anesthesia. After full anesthesia was reached  recordings of anesthetized leucistic axolotl processed with a wide band-pass filter (first part) and a narrow band-pass filter (second part). Note the pronounced noise reduction when processing with a narrow band-pass. Video relates to Fig. fH , thereby validating the accuracy of the EVM method. One-way ANOVA showed that EVM-acquired fH differed statistically significantly between conditions  . A Tukey Honestly Significant Difference post hoc test revealed that resting fH was statistically lower than fH following exercise and during anesthesia , but fH levels at exercise and anesthesia were not statistically significantly different from each other [fH  vs. fH : p = 0.33].Benzocaine has a well-known tachycardiac effect in axolotls ; therefofH Fig. k, n, q. esia) , quad-high definition , full-high definition with high speed , full-high definition , high definition , and video graphics array . Subsequent to EVM, all video formats allowed for a clear heartbeat signal detection and fH measurement Additional file 4. Effect of spatiotemporal resolution on Eulerian video magnified material. Eulerian Video Magnified (color) recordings of anesthetized leucistic axolotl at six different spatiotemporal resolutions. All resolutions allowed for heartbeat signal magnification and rate detection. Video relates to Fig. fH via movements of the front limb in a dark brown wild-type axolotl. A large wild-type axolotl  was anesthetized in 200 mg/l benzocaine, video recorded . Therefore, we wanted to test the applicability of EVM-based motion magnification to detect fH needs to be monitored in multiple animals at the same time with a limited number of cameras and with non-optimal light and background settings, we used the same six leucistic animals as in the rest/exercise/anesthesia experiment. The six axolotls were housed in individual plastic aquaria and placed on a gray linoleum background. Following 48 h of fasting and 24 h of undisturbed rest, a video recording containing all six animals was performed  ± 95% CI = 22.27 ± 4.24 BPM; Fig. t test showed that resting heart rate was not significantly different from previous individual recordings of the same animals (p = 0.99).To mimic an experimental setup in which Additional file 6. Heartbeat magnification in multi-animal setup using Eulerian video magnification. Eulerian Video Magnified (color) recording of six leucistic axolotls at rest. Video relates to Fig. fH detection in a more active ectotherm than the axolotl, we used conscious, unrestrained zebrafish [Danio rerio ]. Forty-five adult (> 4 months of age) mixed sex, wild-type zebrafish  were housed in a 200-l aquarium with fully oxygenated water (90 l) at 27.6 °C. Following 36 h of fasting and 24 h of undisturbed rest to minimize post absorptive heart rate changes and disturbance, video recordings were performed with a remotely controlled camera  ± 95% CI = 127.57 ± 7.87 BPM).In order to test if EVM allowed for Additional file 7. Heartbeat magnification in conscious, unconstrained zebrafish using Eulerian video magnification. Eulerian Video Magnified (color) recording of wild type conscious and unconstrained adult zebrafish. Although the fish is moving slightly during the recording resulting in an increased level of noise particularly in the caudal part of the fish, a heartbeat signal can be extracted from the ventral thoracic surface. Video relates to Fig. Homo sapiens Linnaeus, 1758)  and repeated the procedure described above on the throat (PWV in the left arteria carotis communis) , we first examined the low pressure, low PWV vascular system of the amphibian axolotl. An anesthetized leucistic axolotl  was positioned on the side Fig. a and recAdditional file 8. Pulse wave transit time detection in low blood pressure animal using Eulerian video magnification. Eulerian Video Magnified (color) recording of one anesthetized leucistic axolotl. Pulse wave propagation along the dorsal aorta can be detected at different positions allowing for a pulse transit time measurement. Video relates to Fig. Additional file 9. Pulse wave transit time detection in high blood pressure animal using Eulerian video magnification. Eulerian Video Magnified (color) recordings of resting human being. Dual trace video. Original video to the left and color magnified video to the right. First part shows color magnification on the throat demonstrating pulsations in the arteria carotis communis, second part shows color magnification on the upper arm demonstrating pulsations in the arteria brachialis. Pulse wave propagation along the arteries can be detected at different positions allowing for a pulse transit time measurement. Video relates to Fig. Lacerta agilis Linnaeus, 1758)  and mean respiration rate (94.83 respirations per minute) could be measured over multiple cycles at the level of the heart and the lungs respectively ] that eventually lifted its head up   ] can be directly observed both by incubation in ovo with a window in the eggshell or in ex ovo cultures in which the eggshell is completely removed . Post hoc tests using the Bonferroni correction (α = 0.05/12 = 0.0042) showed that SNR was statistically significantly larger than 1 for the heart measurements on intact eggs at embryonic day 2.38 (p = 0.0014), 3.0 (p < 0.0008), 3.5 (p < 0.0008), and 4.0 (p = 0.0013) , 3.5 (p = 0.0020), 4.0 (p = 0.0026), and 8.0 (p = 0.0038)  = 0.84, p = 0.41], but there was a statistically significant different in fH between different embryonic time points . Six of the selected time points matched embryonic days for which fH has previously been reported . Bonferroni-corrected (α = 0.05/6 = 0.0083) t tests showed that fH for embryos in intact eggs were statistically significantly different from reference values at embryonic day 3.5 (p < 0.002), 4.0 (p < 0.002), and 9.0 (p < 0.002).Development of embryonic domestic chickens . Although the measurements acquired with EVM of the video of a giraffe with the head down and up in this study support this observation ] [Lampornis clemenciae ] [Suncus etruscus ] , and the African forest elephant [Loxodonta cyclotis ] vocalize and communicate with infrasound with fundamental frequencies typically in the range of 14–35 Hz with sound pressure levels as high as 103 dB (re 20 μPa) at 5 m from the source   to 110 k, 1758)] . Sound i, 1829)]  and 1511, 1822)] , could ie source . Infrasoe source , age is e source , 57, ande source . It has e source . Both mee source , 62, thifH, respiration rate, pulse wave velocity, and infrasound vocalization-related vibrations from videographic material in a range of experimental setups. The method does not generate a signal where there is none, but enhances rhythmic signals, such as the heartbeat, relative to the noise level, so an inapparent signal can be localized, visualized, and recorded. The EVM methods work across a wide range of video qualities and spatiotemporal resolutions. Optimally, test objects are stationary, but movements out of the frequency range of the signal of interest can be acceptable. The importance of physiological measurements from undisturbed animals in their natural environment is becoming ever clearer in the fields of experimental biology and ecophysiology, which may in some cases limit the applicability of instrumentation of animals with recording devices. In this respect, EVM can, after a thorough validation with other techniques, provide a tool to prospectively or retrospectively extract signals of interest from a distance using simple inexpensive video recording equipment.In this methodology study, it is demonstrated how EVM can be used to acquire 2 surface area with regular water changes and a 12 h:12 h light to dark cycle. Animals were fed every second day with protein-enriched trout pellets. Axolotl anesthesia was obtained using 200 mg/L ethyl-4-aminobenzoate. Zebrafish were obtained from a commercial breeder  and were housed initially 61 animals together in a large 200-l aquarium with environmental enrichment via grating, brick, and stones; stocking density at the time of recording was 45 fish in the 200-l aquarium. Aquarium air stones provided aerated water, a thermostatic heating system set at 28 °C providing a measured temperature of 27.6 °C, and pump  with a 20-cm water depth and 4500-cm2 surface area with regular water changes and a 12 h:12 h light to dark cycle. Animals were fed daily with JBL NovoBel . Embryonic chicken experiments were performed in the USA (NY) where Institutional Animal Care and Use Committee (IACUC) approval does not apply for embryonic chickens. Eggs were obtained from local commercial breeder and were incubated with rocking at 37.5 °C in a water-saturated environment. All chicken embryos were terminated at embryonic day 9. The embryonic mouse experiment was performed in the USA (NY) where mice were bred at the lab animal facility under IACUC protocol #2008-0011. Mouse anesthesia was obtained by isoflurane inhalation (4% isoflurane in pure O2 for induction of anesthesia and 1.5% isoflurane in pure O2 for maintaining anesthesia), and the adult mouse was euthanized under anesthesia directly after video recording.Animals used for laboratory-based experiments in this study were the Mexican axolotl, the zebrafish, embryonic chicken, and mouse. Axolotl and zebrafish experiments were performed in Denmark and were approved by the Danish National Animal Experiments Inspectorate . Axolotls were obtained from a commercial breeder  and were housed individually in plastic containers with a 10-cm water depth and a 930-cmAll video material from lab-based experiments was recorded using the built-in camera in a standard smartphone  or a GoPro Hero 4  compact camera applying spatiotemporal resolution settings as specified in Table Video material that was not produced with the intention of EVM processing was acquired from external online sources. The sand lizard video was produced by Ben Locke and was located on Flickr ; the girUltrasonography on axolotl and embryonic mouse was performed with a Vevo 2100 system  with a 21-MHz transducer (MS250).Ex vivo micro-CT imaging of a contrast agent  perfused axolotl was performed using a Scanco Medical XtremeCT system  with 1500 projections/180° and an isotropic voxel size of 41 μm, an X-ray tube voltage of 59.4 kVp, an X-ray tube current of 119 μA, and an integration time of 132 ms. In in vivo micro-CT imaging of embryonic chicken, an ex vivo imaging of embryonic mouse was performed using a eXplore CT 120  system with 1200 projections/180° and an isotropic voxel size of 25 μm, an X-ray tube voltage of 80 kVp, an X-ray tube current of 30 μA, and an integration time of 30 ms. For the in vivo embryonic chicken scans, an endoscopic camera (Qyuhe Mini USB Endoscope) was positioned in such a way that the egg surface could be viewed without the endoscope overlapping the trajectory of the X-ray beam of the micro-CT scanner. A flashlight placed directly at the end of the egg provided light for candling and heat during the micro-CT acquisition. Prospective gating was applied via custom-made peak detection software processing the real-time EVM-enhanced signal from the endoscope and signaling the micro-CT system a 5 V transistor–transistor logic false ECG pulse via a Bayonet Neill–Concelman connector.The Matlab source code to perform EVM was released along with the original report of the procedure , and canStable camera. A stable camera setup is all-important when recording videographic material for EVM processing. A moving camera results in a moving video frame, resulting in magnification of movements of all surfaces in the recording. Voice-operated cameras or delayed recording after touching the camera can be useful.Constant background. The magnification technique does not discriminate on objects in focus and those in the background. Therefore, the background in the video should optimally be non-moving and preferably uniform in color.Reflections, shadows, and light source. Reflection and shadows can interfere with EVM processing. If the experimental setup allows for it, reflections and shadows should be kept at a minimal level by adjusting the light source and structures in the vicinity of the scene. Some light sources oscillate with the utility frequency of the power grid, usually 50 or 60 Hz depending on location. For EVM processing in this frequency range, non-oscillating light sources should be used.Non-moving animal. Eulerian video magnification is easiest to implement on non-moving animals. However, if movements appear at a frequency out of the frequency range of interest . When trying to acquire an analog signal with a digital recorder, the frequencies that can correctly be obtained need to be below the Nyquist frequency, i.e., half of the sampling frequency to avoid aliasing . For example, a video recorded at 30 FPS one can only correctly magnify vibrations with a frequency < 15 Hz. Vibrations at higher frequencies may still be magnified so that they are visible on the magnified video, but frequency detection is unreliable in an under-sampled system.Spatial resolution of the video and color depth. High-resolution, large color depth videos are desirable when trying to magnify subtle color changes or faint vibrations. Still, relatively low-resolution videographic material may still be useful [e.g., the low-resolution recording (640 × 480 px2) in Fig. Distance to object and optics. Optimally, the object of interests should be close to the camera to detect small color changes and vibrations. Optics such as telescopic lenses can be used.View point. It is important to consider the camera viewpoint of the object of interest. Some signals may be best observed from specific angles . In some cases, the experimental setup may dictate possible viewpoints that then need to be tested for the applicability with EVM.Duration of video. Eulerian video magnification is a relatively processor demanding technique; therefore, the duration of videographic material should be limited to avoid unmanageable data acquisition.The most important step in the EVM procedure is the acquisition of an input video of adequate quality for subsequent processing. A list of prioritized considerations in the acquisition of videographic material for EVM or evaluation of non-EVM-dedicated material for the procedure is provided below:t tests  were used to test for statistically significant differences between two groups. Relevant types of ANOVAs  were used for omnibus testing of statistically significant difference between more than two groups. For post hoc tests of difference between groups, Tukey Honestly Significant Difference tests or Bonferroni corrected t tests were used. Significance level (α) was set at 0.05 unless specified in the case of the Bonferroni correction. For all statistical tests, p value was reported as actual number unless it was smaller than α/5 in which cases it was reported as p < α/5. Error bars in figures represent 95% confidence intervals.Relevant types of Additional file 5. Heartbeat magnification in colored animal using Eulerian video motion magnification. Eulerian Video Magnified (motion) recording of one brown wild type axolotl axolotl. Dual trace video. Original video to the left and motion magnified video to the right. Note subtle movements of the front limbs in relation to the heartbeat. Video relates to Fig. Additional file 11. Interactive two-phase model of embryonic chicken heart at day 7 of development. Three-dimensional interactive model of beating embryonic chicken heart at day 7 of development in two phases: Ventricular end-diastole and ventricular end-systole. The interactive PDF file should be viewed in Adobe Acrobat Reader 9 or higher. To activate the 3D feature click the model. Using the cursor it is now possible to rotate, zoom, pan the model, and in the model tree all segments of the model can be turned on/off or made transparent. The model tree is a hierarchy containing several sub layers that can be opened (+). Model relates to Fig. Additional file 13. Eulerian video magnification of vibrations related to infrasound vocalization in Asian elephants. Overview of scene and magnified video recordings of three elephants. First part of the video provides an overview of the scene via an aerial photograph (used with permission from COWI) and by a three-dimensional reconstruction of the scene. In the second part four synchronous recordings of vocalizing elephants  from CAM 1–4 that have been video magnified (motion) are displayed. Note that the recordings from each camera have a sound trace in which upper harmonics of low frequency vocalization are audible. Video relates to Figs. Additional file 14. Eulerian video magnification of vibrations related to infrasound vocalization in Asian elephant. Magnified video recording of one elephant close to camera. Video magnified recording of vocalizing elephant . Vocalization results in pronounced vibrations of the elephant (4–12 s in the video). Note that the video has a sound trace in which upper harmonics of low frequency vocalization are audible. Video relates to Fig.


Optical view trocars provide an acceptable level of safety for port entry during minimally invasive surgery. Nevertheless, this technique is not exempt from potential complications. These can range from a minor subcutaneous insufflation that will not require any further treatment to abdominal aorta injuries. A 5 mm optical port was used in the supraumbilical area to gain entry into the abdomen (Video A 52‐year‐old male presented with uncomplicated right inguinal hernia. A robot‐assisted transabdominal preperitoneal repair was planned. The patient had no past medical or surgical history besides the hernia. BMI is 28.72 kg/men Video . After tNone declared.RB: drafted the article, collected the data and images, and edited the video. MM: critically revised the video and manuscript. PF: designed the work, critically revised and approved the final version.VideoClick here for additional data file.


Aggregation of lipids on the tear surface due to saturation was not as significant as it was for meibum. Although the surface pressure/area isotherms for TFL were similar for meibum, differences in rheology and phase transition parameters warrant the study of both.Elevated levels of acyl chain saturation of meibomian lipids are associated with enhanced tear film (TF) stability in infants to shortened TF breakup time with meibomian gland dysfunction. Thus, the effect of saturation on the surface properties of human TF lipids (TFLs) using a Langmuir surface balance and Brewster angle microscopy was studied. Lipid phase transitions were measured using infrared spectroscopy. The raise in the % of saturation resulted in thicker, and more elastic films at π = 12 mN/m, with the effects being proportional to the saturation level. At the same time, at lower (≤10 mN/m) π, the raise in saturation resulted in an altered spreading and modified structure of TFL layers. The strong impact of saturation on TFL surface properties correlated with a saturation induced increase of the TFL acyl chain order, phase transition temperature, and lipid–lipid interactions. The native TFL order and π A thin film of lipids, called the tear film lipid layer (TFLL), covers the surface of the tear film (TF) and serves important functions. The TFLL ensures that the TF has a low surface tension, and an immobile air/TF interface that opposes the outflow of aqueous tears in an open eye ,2,3,4,5.Relationships between TFLL composition, structure, and function could provide insight into the etiology of dry eye. One measurement of lipid structure is termed ‘order,’ which is indirectly related to lipid fluidity that has an additional dynamic component. It is interesting that tear film stability and meibum lipid hydrocarbon order both increase in the order: Meibum from donors without dry eye between the ages of 0 and 25 years old ,19 < meiThe phase transition temperature strongly correlates with lipid order. Lipid unsaturation, the topic of the current study, is the major factor that contributes to lipid disorder and the phase transition temperature ,27,28. OAs the composition and physical properties of TLs are different than that of meibum (above) and saturation contributes to the physical properties of meibum ,27,28,321H NMR spectrum of human tear lipid was typical of that collected on a 700 mHz spectrometer  with saturation, r = 0.971, 0.969, 9.87, respectively. The order of TLs was significantly 35% larger, 42 ± 2% trans compared with meibum collected from the same individual, 31 ± 1% trans.The lipid phase transition temperature for TLs increased with saturation, as expected, from about 30 °C to 51 °C b. Lipid turation a. Above turation e, Δ enthturation c, and Δ turation d increasR) of the complex dilatational modulus for the entire frequency range and a decrease in the tangent of the phase angle. The latter indicates an increase in the contribution of the elastic modulus vs. the contribution of the viscous modulus with saturation. These changes with saturation indicate that the phase coexistence and organization of tear lipid films changes with saturation. Cole–Cole plots confirm that there are at least two processes contributing to the relaxation transients. The Maxwell rheological model equations are summarized in Saturation sequentially increased the maximum surface pressure (at 10% surface area), indicating increased molecular packing density achieved at completion compression . SaturatIn summary, an increase in saturation increases the elasticity and maximum surface pressure and changes the surface phases and phase transitions of the tear lipid layers. An increase in saturation >50% elevates the tear film’s elasticity, but modifies the spreading and results in more heterogeneous layers over a broad surface pressure range. Saturation does not significantly affect the surface properties of films formed by human tears below a surface pressure of 8 mN/m.cis conformation of the double bond, the chains become straighter, allowing for more contact between hydrocarbon chains and greater Van der Waals interactions. Saturation induced stronger lipid–lipid interactions are evident in a linear increase in the change in phase transition enthalpy and entropy with saturation. Saturation also increases the cooperativity of the phase transition. This may be because the system becomes more homogeneous with less of a difference between saturated and unsaturated moieties. One of the major findings of our study is that TL saturation, up to 20% that of native meibum, increased the phase transition temperature and order of the hydrocarbon chains. The correlation between the hydrocarbon order and phase transition temperature has been firmly established ,27,28,32Differences were found between the lipid phase transition parameters of lipids extracted from the tips of Schirmer’s strips and the stems. The differences may be due to tear lipids bound to soluble proteins that migrate up the Schirmer’s strip. Differences between TL extracted from tips and stems of Schirmer’s strips have been characterized . StrongeIt is interesting that a small 1% to 4% increase in saturation did not significantly influence TL rheology, which highlights the resiliency of the TFLL.As indicated, the majority of the lipids in tears are from meibum, but there are significant compositional differences between tear lipids and meibum lipids that could influence the structure, rheology, and function of the tear film lipid layer. Saturation affected tear lipids more significantly than meibum lipids  as the sThe phase transition temperature and order of tear lipids that were catalytically saturated was not significantly different at the same level of saturation compared with meibum lipids that were catalytically saturated. Therefore, the differences in rheology between tear and meibum lipids at a given saturation level are not due to the strength of lipid–lipid interactions, but rather other factors, such as the non-lipid/non-meibomian compounds that come from aqueous tears that are unaffected by catalytic saturation.max would be greater for TLs compared with ML as observed. The film surface compressional modulus, Cs−1, at a given surface pressure occurred over a smaller range, 11 to 12 mN/m, compared with 8 to 80 mN/m for meibum [−1 dependencies indicate the surface pressures at which significant reorganization of the surface film takes place in the course of the film compression. Thus, TL lipids reorganize over a tighter range of surface pressure compared with meibum [As the native TL order was significantly 35% greater than native meibum collected from the same individual, one would expect from the current study that πr meibum . The infh meibum . There wAmong the many factors discussed in the Introduction, saturation could contribute to or be a marker for differences in lipid order and tear film stability, especially between the ages 0 and 25 years and among individuals who are susceptible to severe dry eye who have had hematopoietic stem cell transplantation, as elevated saturation levels for both cohorts have been documented. Indeed, saturation plays a significant role in ordering lens, retinal, muscle, and a variety of synthetic lipids ,27,28,32max were greater in TLs than meibum collected from the same individual. Aggregation of lipids on the tear surface due to saturation was not as significant as it was for meibum. Although the surface pressure/area isotherms for TLs were similar to ML, differences in rheology and phase transition parameters warrant the study of both.In conclusion, saturation increased TLs order, phase transition temperature, lipid–lipid interactions, elasticity, and maximum surface pressure and changed surface phases. Lipid order and πThe materials, diagnosis of normal status, collection and extraction of lipid from meibum, and catalytic hydrogenation sections were identical and copied from .Silver chloride windows for infrared spectroscopy were obtained from Crystran Limited, Poole, United Kingdom. Platinum (IV) oxide was obtained from the Sigma Chemical Company . Unmarked Schirmer’s strips without ink ruling were purchased from Alcon Laboratories .Tears were collected on 54 Schirmer’s strips from each eye of a 63-year-old Caucasian male in the morning for 1 month. The donor status was normal as his meibomian gland orifices showed no evidence of keratinization or plugging with turbid or thickened secretions and no dilated blood vessels were observed on the eyelid margin. The donor did not recall having dry eye symptoms and did not wear contact lenses. Written informed consent was obtained from all donors. Protocols and procedures were reviewed by the University of Louisville Institutional Review Board as well as the Robley Rex Veterans Affairs Institutional Review Board. All procedures were in accordance with the Declaration of Helsinki. Protocols and procedures for the current retrospective study were approved by the University of Louisville Institutional Review Board . The leading tip (5 mm) of the Schirmer’s strip was bent at a 90° angle and subsequently placed over the edge of the lower eyelid for 5 min. The lower portion (5 mm) of the strips was cut off and the upper and lower portions were each placed in a vial filled with argon to prevent oxidation. Care was taken not to contaminate the lower end of the test strip with finger lipids.3 and then again with benzene instead of methanol. The CHCl3 and benzene lipid extracts were added to the methanol lipid extract and the solvents were evaporated under a stream of argon. CDCl3 (1 mL) was added to each sample for NMR analysis and catalytic hydrogenation.Lipids were extracted from the two pools of 54 Schirmer’s strips that were placed in the two glass vials using 5 mL of methanol that had been bubbled with argon for 5 min. The strips and methanol were sonicated with a Sonifier1 cell disrupter microprobe  three times for 15 s each with a 2 min cooling period between sonication. The methanol was decanted and centrifuged at 10,000 rpm for 15 min to remove methanol insoluble impurities. The methanol was decanted again with care not to disturb the pellet. The steps above were repeated using CHClHalf of each of the pooled tear lipid samples was decanted to be catalytically hydrogenated. Saturated tear lipid was prepared as for sphingomyelin ,37. The 1H{13C/15N} 13C-enhanced cold probe . Spectra were acquired with a minimum of 250 scans, 45° pulse width, and a relaxation delay of 1.000 s. All spectra were obtained at 25 °C. The TMS resonance was set to 0 ppm. Commercial software  was used for spectral deconvolution and curve fitting. Spectral data were acquired using a Varian VNMR 700 MHz NMR spectrometer  equipped with a 5 mm 3 was applied to a AgCl infrared window. The solvent was evaporated under a stream of argon gas and the window was placed in a lyophilizer for 4 h to remove all traces of solvent. Infrared spectra were measured using a Fourier transform infrared spectrometer, Nicolet 5000 Magna Series . Lipid on the AgCl window was placed in a temperature-controlled infrared cell. The cell was jacketed by an insulated water coil connected to a circulating water bath, modelR-134A . The sample temperature was measured and controlled by a thermistor touching the sample cell window. The water bath unit was programmed to measure the temperature at the thermistor and to adjust the bath temperature so that the sample temperature could be set to the desired value. The rate of heating or cooling (1 °C/15 min) of the sample was also adjusted by the water bath unit. Temperatures were maintained within ±0.01 °C. Exactly 100 interferograms were recorded and averaged. Spectral resolution was set to 1.0 cm−1.Lipid phase transitions were measured as described previously . Approxi2 stretching band near 2850 cm−1 (trans and gauche rotamers (lipid order) in the hydrocarbon chains as described [2 stretching band near 2850 cm−1 and Tc is the phase transition temperature. Hillslope is the relative cooperativity. The broader the phase transition, the smaller the value for the hillslope. This term describes how the order of a lipid influences that of neighboring lipids. The data for the percentage of trans rotamer were used to calculate the phase-transition enthalpy and entropy from the slopes of Arrhenius plots.The frequency of the symmetric CHescribed . Lipid p2, volume 100 mL  by the Wilhelmy wire probe method  [−1, at a given surface pressure was calculated from the π/A compression isotherm as described [Surface pressure–area (π-A) isotherms were measured using Langmuir surface balance µTrough XS, area 135 cm01 mN/m) ,39,40. Tescribed ,42.0, of 15 mN/m. Then, the lipid film was instantaneously and slightly contracted with a compression step, ∆A/Ao = 5 ± 1% . As discussed elsewhere [In order to gather information about the dilatational viscoelasticity of meibum/tear films that were catalytically saturated, the relaxation of the surface pressure was monitored after a small rapid compression deformation was applied to the surface film as described in detail in ,43,44. Flsewhere ,42,43,44IM vs. ER) as described [The Fourier analysis of the relaxation transients was performed as previously described ,42,43,44escribed ,42,43,44escribed ,42,43,44p, were obtained from a critical value table of the Pearson product-moment correlation coefficient. A value of p < 0.05 was considered statistically significant.Curves were fitted using Sigma plot 10 software  and the confidence levels,


Nursing home residents often suffer from multi-morbidities and geriatricsyndromes leading to lower quality of life or mortality. Oropharyngeal dysphagia(OD) and malnutrition are profound conditions in this complex profile ofmulti-morbidities and are associated with deprived mental –and physical healthstatus, e.g. aspiration pneumonia or dehydration. This study aimed to assess theassociation between OD and malnutrition in Dutch nursing home residents.Data for this cross-sectional study were obtained from the annual NationalPrevalence Measurement of Quality of Care (LPZ).The National Prevalence Measurement of Quality of Care was conducted inNursing Homes in The Netherlands.Participants were nursing home residents age 65 or older and admitted topsychogeriatric- or somatic wards.The measurements were taken by trained nurses from the participating nursinghomes. Anthropometric measurements and unintended weight loss (%) were assessed todetermine nutritional status . OD was assessed by means of astandardized questionnaire assessing clinically relevant symptoms of OD such asswallowing problems or sneezing/coughing while swallowing. Cox regression wasapplied to assess the association between malnutrition and clinically relevantsymptoms of OD in older Dutch nursing home residents.Approximately 12% of the residents suffered from swallowing problems and 7%sneezed/coughed while swallowing liquids or solid foods. Approximately 10% of theresidents was malnourished. Residents with OD symptoms were more oftenmalnourished compared to residents without OD symptoms. Approximately 17% of theproblematic swallowers were concurrently malnourished. Increased risk formalnutrition was found in residents suffering from swallowing problems , as well as in residents that sneezed/ coughed while swallowing . Stratification based on wards revealed that problematicswallowers from somatic wards were at a high risk of malnutrition .Clinically relevant symptoms of oropharyngeal dysphagia, such as swallowingproblems and sneezing/coughing while swallowing are associated with increased riskof malnutrition in psychogeriatric and somatic Dutch nursing homeresidents. Oropharyngeal dysphagia (OD) and malnutrition are conditions that result inlower quality of life and that place people at high risk for co-morbidities andmortality. OD is considered a new geriatric syndrome , and freOD and malnutrition complicate care in older nursing home residents in view ofassociated health complications, co-morbidities and a deprived mental health status.When OD and malnutrition are underestimated, unrecognized  or In order to diagnose OD, the volume-viscosity swallow test (V-VST) is currentlyrecognized as the gold standard ,howeverSimilar to the diagnosis of OD, a variety of definitions, measurements and toolsto determine nutritional status are applied  since thTreatments in malnourished residents suffering from OD are of compensative orrehabilitative nature and include e.g. diet modifications, nutritionalsupplementation, oral-motor therapy, postural techniques and/or facilitationtechniques . In geneOverall, prevalence rates found for OD and malnutrition are inconclusive andthere is some evidence that mortality is even more prevalent in coexistingoccurrence of OD and malnutrition .HoweverData were obtained from Dutch nursing home residents that participated in theannual cross-sectional National Prevalence Measurement of Quality of Care (LPZ)measurement rounds of 2016 or 2017. The study population included residents of 65years or older, living in somatic- and psychogeriatric wards of nursing homesacross the Netherlands. Data of residents that received palliative care at the dayof the measurements were excluded. Detailed information on the study design of theLPZ is available in the study by van Nie-Visser et al. (2013) .Approval for the LPZ was given by the Medical Ethical Committee of MaastrichtUniversity and the Academic Hospital Maastricht . Participation was voluntary and none of the participating residents,nurses, nursing homes or care institutions received financial compensation.Data on resident characteristics , and primary outcome measures  werecollected on a pre-set measurement date. Trained nurses from different wardswithin the nursing home collected the data and entered and submitted the dataelectronically .The care dependency scale (CDS) is a validated assessment tool to indicateresidents’ needs and dependency status. The CDS consists of 15 items, each ratedon a five-point Likertscale. A reduced CDS indicated a higher care dependency ofthe resident  .The standardized questionnaire of the LPZ was established based on literatureand consultation of experts  and included two questions onclinically relevant symptoms of oropharyngeal dysphagia. Questions asked were:“Does the client have swallowing problems?” and “Does the client sneeze or cough while swallowing food or liquids?”.2, orwith a reduced BMI (a BMI below 20 kg/m2 in residentsaged 65–70 years or a BMI below 22 kg/m2 in residentsage 70 or older) in combination with recent unintended weight loss (>5% overthe past 3 months or >10% indefinite of time).Malnutrition in the nursing home residents was indicated based on theoperational definition for malnutrition in older people of the European Societyfor Clinical Nutrition and Metabolism (ESPEN) . Data onWith a multiple-choice question, the nurses could indicate which nutritionalinterventions the residents received. Nutritional interventions included forexample nutritional supplementation and enriched snacks, but also adjustments offood consistency and mealtime-ambiance or referral to a dietician. For residentswith symptoms of OD, additional questions on meals and beverage consistencies andreferral to a speech-language therapist were incorporated: “Does the clientreceive mashed meals or thickened beverages because of swallowing problems?”  and “Is the client supervised by a speech-language therapist becauseof swallowing problems?” . 2 or bodyheight < 108 cm) were eliminated. Of residents that participated in bothmeasurement rounds, 2016 and 2017, only the data of 2017 were included. Prior toanalysis, numerical data on BMI and weight loss were recoded into a dichotomousvariable on malnutrition based on the ESPEN definition for malnutrition.Independent sample T-tests and Chi-square tests were conducted to check fordifferences between groups. To assess the association between OD and malnutritionin older nursing home residents, the crude and adjusted prevalence ratios (PR)were subtracted from Cox regression to prevent overestimated associations fromlogistic regression . Normality of the data was determinedwith QQ-plots. Data of residents with missing values for primary outcomes oroutliers  and a mean CDS of 42.4 (SD 16.6).Significantly higher mean CDS was found among somatic residents as compared topsychogeriatric residents (p< 0.001). No differences were found between the twostudy rounds for prevalence rates for malnutrition  or for prevalence rates for sneezing/ coughing while swallowing. The prevalence of residents with swallowingproblems was higher (p=0.017) in 2016 (13.0%) compared to the prevalence ofresidents with swallowing problems in 2017 (11.1%).The study population consisted of 6349 older residents from Dutch nursinghomes. Almost two-thirds (66.0%) were residents from the psychogeriatric wards andthe remaining residents (34.0%) were admitted to somatic wards. The majority wasfemale (70.2%) with a mean age of 84.5 years (SD 7.5), a mean BMI of 24.8kg/mApproximately one out of eight residents suffered from swallowing problems(12.1%) and one out of fourteen residents sneezed/coughed while swallowing liquidsor solid food(6.9%). If somatic ward residents who suffered from stroke wereexcluded, the prevalence of residents with swallowing problems was higher(p=0.025) in psychogeriatric wards (11.3%) compared to somatic wards (9.2%). Oneout of ten residents was malnourished (10.3%) and malnutrition was more often (p =0.002) seen in psychogeriatric residents (11.1%) compared to somatic residents(8.7%). Residents with swallowing problems were more often malnourished compared toresidents without swallowing problems, with almost one out of every fiveproblematic swallowers being malnourished (17.2%). Almost half of the problematicswallowers indicated additional problematic sneezing/coughing in the act ofswallowing (46.9%). Nearly all residents that indicated sneezing/coughing whileswallowing had overall problems swallowing (82.2%). As shown in Among malnourished residents, approximately one out of five was suffering fromswallowing problems (20.2%) and one out of ten was sneezing/coughing whileswallowing foods or liquid beverages (10.2%).In comparison to non-malnourished residents , theaverage CDS was lower (P<0.001) in malnourished residents .More than two-thirds of the residents were diagnosed with dementia (65.6%) andnearly half was diagnosed with disease of the circulatory system (44.1%). DementiMalnourished residents with clinically relevant symptoms of OD were mostlyreferred to a dietician (57.7%), or received energy (E+) and protein (P+)enriched- diets (29.2%) and/or snacks (48.5%). The majority of the residents with clinically relevant symptoms of OD werereferred to a speech/language – therapist. A 74.6% of the residents that sufferedfrom swallowing problems and a 79.2% of the residents that were sneezing/coughingwhile swallowing were referred to a speech/language – therapist.Univariate analysis  showedaIn stratified analysis increased risks for malnutrition amounted to 1.9  and 1.7  among residents withswallowing problems at psychogeriatric and somatic wards respectively. Residents at psychogeriatric wards and with sneezing/coughing while swallowingdid also show an increased risk of malnutrition .As shown in In stratified analysis, increased risks of malnutrition amounted to 1.4  and 1.9  among residents withswallowing problems at psychogeriatric and somatic wards respectively.This cross sectional prevalence study showed prevalence figures of oropharyngealdysphagia and malnutrition among older Dutch nursing home residents and revealedsignificant associations between oropharyngeal dysphagia and malnutrition amongthese nursing home residents.The overall prevalence of OD in this current population was lower compared toreported prevalence numbers from previous studies , 36. In Nevertheless, even without instrumental visuo-perceptual assessment methods,prevalence rates of OD up to 20.2% were found in the current study amongmalnourished residents. This finding is in line with the results of a similar studyby Poisson et al. (2016) among hospitalized older people . Almost The prevalence of malnutrition in this study population is also relatively lowas compared to prevalence rates of malnutrition in the literature , 24. HowInterestingly, Poisson et al. (2016) also found an association betweenmalnutrition and oral self-care dependency. Similar results were found in thecurrent study, where lower average care dependency scores (CDS), meaning higher caredependency, were found in residents suffering from OD and in malnourishedresidents.With regard to clinically relevant symptoms of OD, subjective swallowingproblems were often accompanied by sneezing/coughing in this study. However, someresidents without subjective swallowing problems indicated problems with eating dueto sneezing/coughing while swallowing, probably also related to dysphagia as it isknown that coughing during oral intake is related to penetration or aspiration. In the The group of residents admitted to somatic wards is a relatively small group,approximately one third, of the total population. The majority of residents isadmitted to psychogeriatric wards, with dementia as the most frequently occurringclinical diagnosis. A previous study that was conducted in Finnish older nursinghome residents revealed two - and three - fold risks for malnutrition due todementia  and swallowing problems . SwallowMore specific reference data from the literature on differences betweenpsychogeriatric and somatic wards in nursing homes are lacking at the moment sincemainly in Dutch nursing homes these specific distinctions have been made. Inaddition, to compare the findings of the current study to the literature, take intoconsideration the difference between varying statistical methodologies to assessassociations. Previous studies were based on logistic regression, a commonly usedmethod for the assessment of associations, though known to overestimate associations, 35. TheIn the present study nurses reported clinically observed symptoms or complaintsof dysphagia. Another method of swallowing assessment may have produced differentresults although instrumental swallowing assessments such as videofluoroscopy arenot available in Dutch nursing homes and fiberoptic endoscopic evaluation ofswallowing is not possible on such large scale sample sizes of vulnerable nursinghome residents.No conclusion on causality between OD and malnutrition can be drawn from thecurrent cross-sectional study design, however the clear evidence of an associationbetween OD and malnutrition shows the need for more research on this issue.Clinically relevant symptoms of oropharyngeal dysphagia, such as swallowingproblems and sneezing/coughing while swallowing are associated with increased risksfor malnutrition in psychogeriatric and somatic Dutch nursing home residents. Futureresearch is needed to increase understanding and awareness among affected residentsand involved healthcare disciplines to optimize care, tailored to the needs ofpsychogeriatric and somatic residents with OD and malnutrition in Dutch nursinghomes.Author Declaration and Ethical Standards: AvHis employed by Danone Nutricia Research. No other conflict of interests have beendeclared by the authors. We further confirm that any aspect of the work covered inthis manuscript that has involved human patients has been conducted with the ethicalapproval of all relevant bodies and that such approvals are acknowledged within themanuscript.