The binding of Ca2+/Mg2+ to this indicator changes the conformation of human centrin 3, and induces bioluminescence resonance energy transfer (BRET) from Nanoluc to Venus, which changes its emission spectrum about 140%. The dissociation constants of LOTUS-W for Ca2+/Mg2+ are approximately several mM, making it suitable for measuring WH in the household water. With this indicator in combination with a smartphone, we have demonstrated that it is possible to evaluate WH easily and quickly. This novel indicator has the potential to be used for measuring not only household water but also water used in the food industry, etc.Water hardness (WH) is a useful parameter for testing household water, such as drinking, cooking, and washing water. Many countries around the world use pipeline water in their houses, but there is a need to monitor the WH because hard water has a negative impact on appliances. Currently, WH is often measured using chemical dye-based WH indicators, and these techniques require expensive equipment, and trained personnel. Therefore, a low-cost and simple measurement method has been desired. Here, we report LOTUS-W, which consists of a luciferase, Nanoluc, a yellow fluorescent protein Venus, and a Ca However, their concentrations are much lower than Ca2+ and Mg2+. WH is determined based on the total concentration of Ca2+ and Mg2+ in parts per million (ppm) or milligrams per liter (mg/L) [Natural water contains several minerals, such as calcium, magnesium, potassium, phosphate, and carbonate. These minerals are generally added when natural water passes through soils and rocks containing large mineral deposits. Water with high concentrations of polyvalent cations >+1), such as Ca, such asr (mg/L) .2+ and Mg2+ from the hard water by replacing two Na+ [2+/Mg2+. After softening a large quantity of hard water, the exchange medium accumulates a large amount of Ca2+ and Mg2+, and causes the internal degradation of this ion exchange resin that leads to ineffective Ca2+/Mg2+ removal in household water [According to the United States Geological Survey (USGS), domestic water can be categorized into indoor and outdoor uses of household water. Generally, indoor household water is for drinking, food processing, bathing, flushing toilets, washing clothes and dishes, and outdoor uses are for watering lawns, gardens, and maintaining pools. Household water is provided by public suppliers or self-supplied. In the USA, 283 million people nearly 99%) collect their household water from the public water supply system through the pipeline (tap), and 42.5 million people 1%) privately manage their domestic water from fresh groundwater sources ,7. From 9% collec% privateld water ,15,16. TFor the determination of household WH, chemicals such as eriochrome black t, pontachrome black TA, eriochrome-schwartz-T, ethylenediaminetetraacetic acid, and bismuth oxoiodide have been used ,17,18. T2+ and Mg2+, another study from the same group also used this system to detect various salts such as KCl, NaCl, FeCl3, FeSO4, and AlCl3, suggesting that the indicator has low specificity for Ca2+ and Mg2+ [Fluorescent protein-based indicators have been used as a tool for environmental applications such as metals detection in water and soil ,23. Försand Mg2+ . In addiand Mg2+ ,28. Theiand Mg2+ ,30. The and Mg2+ ,32,33. T2+ and Mg2+ are suitable for measuring WH in household waters. In addition, this bioluminescent indicator is able to rapidly detect WH by using a portable device such as a smartphone.Here, we report on the development and application of a bioluminescent protein-based ratiometric WH indicator for the assessment of hardness in household waters. The binding affinities to CaDNA oligonucleotides used for cloning and construction of this indicator in this study were purchased from Hokkaido System Science Co. Ltd. , and are listed in BamHI site and a reverse primer with a SphI site. The cDNA of NanoLuc (Nluc) was amplified with a sense primer with an attached SacI site and a reverse primer with EcoRI. cDNA of human centrin 3 (HsCen3) was amplified from the expression vector magFRET-1  [SphI site and a reverse primer with SacI site. To obtain Venus/cpVenus-HsCen3-Nluc, amplified PCR fragments were digested with the enzymes and inserted into the pRSETB  vector between BamHI and EcoRI sites by four-piece ligations. Construction of Nluc-HsCen3-Venus/cpVenus was performed by replacing each of the genes. Deletion of the C-terminus of the HsCen3 indicator was performed by inverse PCR with primers listed in Venus cDNA and the circularly permuted (cp) Venus series  were cloned from BRAC . These c #50742) , using aEscherichia coli strain JM109 (DE3), and cultured at 23 °C for 69 h in 200 mL LB bacterial growth medium supplemented with 100 µg/mL carbenicillin. Cultured cells were suspended in PBS buffer, and lysed using a French press (ThermoFisher Scientific). The lysate was centrifuged (8000 rpm at 4 °C for 20 min), and recombinant proteins were purified from the supernatant using Ni-NTA agarose affinity columns (QIAGEN), followed by buffer exchange with 20 mM HEPES (pH 7.4) with the desalting column, PD-10 . After lysis, all protein purification processes were conducted on ice to avoid protein degradation. Protein concentration was determined by both the alkaline-denaturation method [WH indicator was expressed in n method ,37 and t2+ and Ca2+ titrations were performed in solutions containing 10 mM MOPS (pH 7.2) and 100 mM KCl at 25 °C. After a full scan of the light emission spectrum from 400–650 nm, the ratiometric value (ratio) was obtained by dividing the acceptor (cpVenus) emission peak (525 nm) by the donor (Nluc) emission peak (455 nm). The dynamic range of the WH indicator was determined based on the ratio in the presence and absence of Ca2+/Mg2+. The dissociation constants (Kd) for Mg2+ and Ca2+ were calculated by non-linear regression analysis of the ratio-based titration curve. Ratios were normalized with a maximum count. Sigmoidal-binding curves were fitted to the data using the Origin7 software  to calculate using the Hill equation.The emission spectra of the WH indicator was measured by adding 5 µM coelenterazine h to 50 nM or 4 nM of protein using a spectrophotometer  or a micro-plate reader . The emission spectra were normalized at the iso-emission point (506 nm) and Mg2B4O7·10H2O, and 30 mM C6H5Na3O7·2H2O [To measure pH dependency, buffers with different pH values were prepared using 30 mM Na3O7·2H2O . The pH 2+ and Ca2+. Li+, K+, Mn2+, Fe2+, Co2+, Ni2+, Cu2+, Zn2+, As3+, Cd2+, and Pb2+ buffers were prepared by using LiCl, KCl, MnCl2, FeCl2, CoCl2, NiCl2, CuCl2, ZnCl2, AsCl3, CdCl2, and PbCl2, respectively, containing 10 mM MOPS (pH 7.2) and 100 mM KCl. Buffers with the same base and no additional metal ions were used as controls. Specificity was determined with 50 nM LOTUS-W by adding 10 mM Mg2+ or 10 mM Ca2+ in the presence of 3 µM (mostly harmful ranges) of metal ions. All assays were performed by using a microplate reader.Purified LOTUS-W was prepared in buffers containing different metal ions with and without MgThe temperature sensitivity of LOTUS-W was analyzed using a spectrophotometer. The temperature of the sample was regulated by a temperature-sensitive electrode. Bioluminescence of a mixture containing 50 nM LOTUS-W and 5 µM Coelenterazine h, at temperatures ranging from 25–45 °C, was measured.2+ and Mg2+ concentrations (mg/L). Previous studies have calculated water hardness using certain equations [The WH scale was defined according to the guidelines of USGS/WHO . Non-carquations ,40. For 4 pixels, ISO: 800, and the F-score: 1.7, outer camera) smartphone was set at an exposure time of 15 ms. Images were taken in a dark box. The color balance  was set manually between 5000K and 5500K. The original RGB images were analyzed using MetaMorph , and Image J. The ratio was calculated from the averaged green and blue intensities, as shown previously [The bioluminescence reaction was performed in 96-well microplates with 50 µL of 4 nM LOTUS-W, 50 µL of WH buffer, and 100 µL of 5 µM coelenterazine h. The Galaxy S8+ The total WH expressed as the concentration of calcium and magnesium ions are equal to the concentration of CaCO WHO/EPS  and the 3, Ca2+, and Mg2+ are 100.1 g/mol, 40.1 g/mol, and 24.3 g/mol, respectively.Here, the molar masses of CaCOLuminescent Optical Tool for universal sensing of voltage (LOTUS-V) [2+/Mg2+ sensing domain human centrin 3 (HsCen3) [Previously, we developed a genetically encoded bioluminescent indicator for monitoring the voltage of a cell membrane called LOTUS-V) . This in(HsCen3)   from Nluc to cpVenus. These candidates were purified and their luciferase activity was characterized by the addition of Mg2+. Based on the screening by dynamic range, we selected cp229Venus∆C10–HsCen3–Nluc (cpV229–HsCen3-NL), because it showed the highest dynamic range of about 28%  for Ca2+ was 2.1 mM  and that for Mg2+ it was 4.7 mM   [To further improve cpV229–HsCen3-NL, 10 additional variants were created by truncation of HsCen3 at the C-terminus A, and thand Mg2+ . From thand Mg2+ C,E, respnt, 1.6) D,F. The to Ca2+) . The dyn2+ and Mg2+ for other metal ions: Li+, K+, Mn2+, Fe2+, Co2+, Ni2+, Cu2+, Zn2+, As3+, Cd2+, and Pb2+. Comparing the metal ions, Ca2+ and Mg2+ induced noticeable changes in the bioluminescence ratio of cpV229N_HsCen3∆C7-NL (2+ and Mg2+.We confirmed the specificity of cpV229N_HsCen3∆C7-NL to Can3∆C7-NL A. Their n3∆C7-NL B,C. Thus2+ and Mg2+ did not fluctuate at different pH values, cpV229N_HsCen3∆C7-NL could be used for WH measurements over a wide pH range. The indicator is hypothesized to be useful in different temperature conditions. For this purpose, we also studied temperature dependency (Next, the pH-dependence of cpV229N_HsCen3∆C7-NL was analyzed between pH 3–11 . As the pendency . The respendency . Thus, cRecently, imaging based on mobile devices has been offered as an attractive analytical technique in biological and environmental monitoring . EspeciaThus, the portable bioluminescence indicator, LOTUS-W, can be set up on-site to measure WH in household waters. The time from sample preparations to the acquisition of the final image was estimated to be within 1–2 min. This is a practical performance time for on-site applications.2+ or Mg2+ in LOTUS-W would need to be optimized by engineering the indicator domain for the practical applicability of this indicator. The combinatorial use of this indicator with different affinities for Ca2+ and Mg2+ has the possibility to compensate for the distortion caused by different affinities for Ca2+/Mg2+ in LOTUS-W, and improve the measurement accuracy. We estimated the testing cost for a single measurement is approximately 10 times higher than current commercially available kits. The higher cost compared to conventional products is an issue that will have to be solved in the future. A simple inspection method along with LOTUS-W and portable devices can be used as a potential tool in various applications of WH.In this study, we constructed a noble BRET indicator LOTUS-W for the detection of WH in household waters. We have demonstrated the BRET ratio of commercially available bottled waters WH on relative quantification methods. A smartphone-based camera was introduced to detect the bioluminescence and reflect its values. This detection system offers to instantly analyze the images of WH effectively by using a digital color image analyzing system . Urbaniz


Patients need to be educated about possible treatment choices in order to make informed medical decisions. As most patients are medical laypeople, they find it difficult to understand complex medical information sufficiently to feel confident about a decision. Multimedia interventions such as videos are increasingly used to supplement personal consultations with medical professionals. Former research has shown that such interventions may have a positive effect on understanding, decision making, and emotional reactions. However, it is thus far unclear how different features of videos influence these outcomes.We aimed to examine the impact of visualization formats and basic navigational options in medical information videos about cruciate ligament surgery on recipients’ knowledge gain, emotions, attitude, and hypothetical decision-making ability.In a between-group randomized experiment (Study 1), 151 participants watched 1 of 4 videos . In a separate online survey (Study 2), 110 participants indicated their preference for a video design. All participants were medical laypeople without personal experience with a cruciate ligament rupture and were presented with a fictional decision situation.P=.005) and procedural knowledge (P<.001) than participants who did not have or use navigational options (n=115). A realistic visualization induced more fear (P=.001) and disgust (P<.001) than a schematic video. Attitude toward the surgery (P=.02) and certainty regarding the decision for or against surgery (P<.001) were significantly more positive after watching the video than before watching the video. Participants who watched a schematic video rated the video significantly higher than that by participants who watched a realistic video (P<.001). There were no significant group differences with regard to hypothetical decision making and attitude toward the intervention. In addition, we did not identify any influence of the visualization format on knowledge acquisition. In Study 2, 58 of 110 participants (52.7%) indicated that they would prefer a schematic visualization, 26 (23.6%) preferred a realistic visualization, 17 (15.5%) wanted either visualization, and 9 (8.2%) did not want to watch a video at all. Of the participants who wanted to watch a video, 91 (90.1%) preferred to have navigational options, 3 (3.0%) preferred not to have navigational options, and 7 (6.9%) did not mind the options.In Study 1, participants who used navigational options (n=36) gained significantly more factual knowledge   Original Paperpreference sensitive, because they should consider individual circumstances and preferences of the patient as well as scientific evidence. Most patients prefer making shared or autonomous medical decisions [In clinical practice, doctors and patients are sometimes faced with a situation that has no clearly optimal treatment. Nonetheless, they have to make a decision on the most-appropriate option. Such decisions are ecisions . Althougecisions . To makeecisions . Severalecisions . In addiecisions  as compaecisions  when theImplementing shared decision making is challenging. Many patients feel that they lack the knowledge to make an informed decision and therefore underestimate the importance of personal preferences and individual experiences ,8. PatieDecision-support tools are increasingly used to support patients who have to make a medical decision. They can be applied before, during, or after a clinical encounter; provide evidence-based information about options, risks, and benefits; and support the decision-making process by helping patients imagine the different options and clarify their personal values . DecisioExperimental studies that explicitly tested the use of decision aids during the informed-consent process found that the aids consistently had a positive impact on knowledge gain -18. OnlyA review by Stacey and colleagues  showed tVideos are often included in many decision-support tools, usually as part of a more complex multimedia intervention. The use of videos as a source of medical information has increased in the past years  in a proIn summary, research suggests that videos can be useful in improving patient education, may regulate emotions, and have the potential to support decision making. However, the videos used in these studies were diverse, which may explain the difference in some aspects of previous findings. Thus far, there has been no research on the potential impact of different designs of video formats. In addition, only a few studies have specifically investigated the influence of different videos on decision making. For patient counseling, it is important to examine which features of educational videos may promote knowledge gain, evoke or avoid negative emotions, and support attitude formation in medical decision making.visualization format is the use of schematic or realistic presentations. A schematic video consists of animated line drawings that present the most-relevant parts or components but omit negligible details. In contrast, a realistic video shows real representations and processes and depicts them in their actual complexity. In the case of a surgery video, a realistic film would show an actual surgery on a real body. Studies in nonmedical contexts have shown that schematic videos led to better learning outcomes [The design of educational videos can differ in many ways. One possible variation for the outcomes ,35 and woutcomes . Howeveruser control. The availability of basic navigational options to stop and skip forward or backward was found to facilitate the learning process in nonmedical domains [Another type of variation in educational videos is the amount of  domains -39. To oHerein, we conducted two studies to compare differently designed videos in a randomized controlled experiment and to examine people’s preferences for particular designs of medical information videos in an online survey.arthroscopic cruciate ligament surgery, which is a common orthopedic surgical procedure and a frequently performed treatment after a cruciate ligament rupture [In our first study, we aimed to compare different visualization formats  and basic navigational options (available or unavailable) in medical information videos in terms of their impact on knowledge, emotions, and attitudes. In addition, we examined the influence of the two abovementioned factors on patients’ decision to undergo one of two medical treatments. As a video topic, we chose  rupture ,41. Anot rupture -44. TherAs the influence of video design on attitude formation and decision making in a medical context has not been examined thus far, we investigated these influences in the form of explorative questions. Additionally, we asked participants for a general evaluation of the video.Previous research has found that schematic videos made learning of complex topics easier as compared to realistic videos -36. Accoarthroscopic cruciate ligament surgery. In contrast to Study 1, which was conducted as a laboratory experiment, Study 2 was performed as an online survey.In our second study, we wanted to learn more about people’s preferences for visualization formats and basic navigational options to complement the results of the first study. As in Study 1, we used the topic This research was performed in accordance with the Declaration of Helsinki and received full approval by the ethics committee of the Leibniz-Institut fuer Wissensmedien .The trial was not preregistered as Study 1 was conducted before trial registration became standard policy at the Leibniz-Institut für Wissensmedien.We conducted a randomized controlled experiment in a laboratory setting. The participants were placed in a hypothetical situation where they would have to confront the possibility of surgery. They watched an educational video and responded to several questionnaires about their knowledge , emotions (fear and disgust), and attitude toward the intervention as well as their decision and certainty of the decision.P=.19; n=151), distribution of gender , occupation , or education . All participants provided written informed consent. The study lasted for approximately 45 minutes and was compensated with 6 Euros.To be able to detect a medium effect size , a sample size of 40 per condition was required. The experiment was performed with 157 laypeople who were randomly recruited from the participant database of the Leibniz-Institut fuer Wissensmedien and invited via email. Registration in this database was voluntary and open to everyone who was willing to participate in empirical studies. Medical or sports students and people working in a medical profession were not invited to participate, because we aimed to include participants who had no or little prior professional knowledge about the topic and wanted to determine how laypeople, in particular, reacted to the videos. In the invitation, potential participants were informed that during the experiment, they might watch a video demonstrating the surgery on a body donor. They were repeatedly informed that they could stop the video at any point without any negative consequences. Of the 157 participants who were initially recruited, 6 participants were excluded: 1 participant had technical problems, 1 had specified a mother tongue other than German, and 4 did not follow the instructions . The remaining 151 participants . Subsequently, they were asked to place themselves in a fictional situation where they had suffered a cruciate ligament rupture in a sports accident and were faced with a choice for or against surgery. They were asked about their decision , certainty of this decision, and attitude toward the potential surgery (premeasures). Thereafter, they were asked to imagine that before the next consultation, their doctor gave them an information brochure about their injury and recommended a video on the possible surgery. At this point, the same information sheet was handed out to every participant. It included general information on the location and function of the cruciate ligament and consequences of an injury and treatment options (surgery or intense physiotherapy). This information was provided in a neutral manner. Patients were also informed that in medical research, thus far, no treatment was found to be clearly better than the others and that each patient had to make an individual decision. This information sheet comprised 679 words. After they finished reading this sheet, the participants watched one of four different videos, depending on their experimental condition. They were randomly assigned to the conditions by the survey program Qualtrics . The videos differed in their visualization format . Attitude toward the intervention was measured using 4 bipolar items on a 7-point scale before and after watching the video [αatt-pre=.70, Cronbach αatt-post=.80). The participants’ hypothetical decision was noted before and after watching the video. On a 5-point scale, they indicated how likely they were to hypothetically undergo surgery in the situation they were given.To measure the participants’ emotions while watching the video, we used the subscales ct Scale . On a 5-certainty regarding the decision, participants were asked in 5 items how certain they felt about their decision for or against the surgery. All responses were given on a 5-point scale, and the items showed acceptable internal consistencies . General evaluation of the video was measured using 4 items. Internal consistency was acceptable (Cronbach αsat=.66). To measure P<.05. The partial eta-squared value was calculated as effect sizes of mean differences.Data analysis was performed using IBM SPSS 22 statistics for Windows . To test for differences between the conditions, we performed analyses of variance, including repeated measure analysis for the pre-post comparisons. We report all data as means and standard deviations. The level of significance was set at The online survey was performed with 110 laypeople  a schematic video, (b) a realistic video, (c) any video without preference for the visualization format, or (d) no video at all. If they answered a, b, or c, they were asked if they would like to have basic navigational options . The survey only consisted of these basic questions and did not include any manipulation, since this study was intended to identify people’s general preferences for medical videos.Chi-squared tests were used to test for differences in frequencies.factual knowledge  or procedural knowledge .In contrast to Hypothesis 1, there were no significant group effects of the visualization format regarding P=.10, procedural knowledge: P=.51). There was a significant difference between the participants who had used the navigational options at least once (n=36) and those who had not used the options at all (n=115). Participants who used the options at least once performed significantly better in both factual knowledge =8.18, P=.005), and procedural knowledge =11.94, P<.001).As per Hypothesis 2, we expected participants who were given basic navigational options to gain more factual knowledge and procedural knowledge than participants without navigational options. However, we did not observe the expected group differences =11.12, P=.001) and disgust =34.14, P<.001) than participants who had watched the schematic video.In support of Hypothesis 3, participants who had watched the realistic video experienced more part.η2=.34, F=75.71, P<.001). In addition, we found no differences between the video types.In support of Hypothesis 4, certainty about the decision was significantly higher after the video presentation  than before the presentation  (P>.09). However, there was a significant pre-post effect on attitude: Participants’ attitude toward the intervention was more positive after watching the video  than before watching the video  =5.27, P=.02).Regarding our explorative analysis, we found no significant group differences for attitude toward the intervention  or certainty about the decision . However, we found that participants who had watched a schematic video rated this video significantly higher  than that by participants who had watched a realistic video  =20.34, P<.001).There were no significant group effects on decision =76.95, P<.001). Regarding the visualization format, 58 participants (52.7%) preferred to watch a schematic video, 26 (23.6%) opted for a realistic video, and 17 (15.5%) did not favor any format =50.36, P<.001). Of those who wanted to watch a video, 91 participants (90.1%) preferred to have basic navigational options, 3 (3.0%) preferred not to have navigational options, and 7 (6.9%) did not care =146.69, P<.001).Of the 110 participants, 101 (91.8%) wanted to watch a video and 9 (8.2%) preferred not to watch any video . ,48. 47,4The use of basic navigational options resulted in better performance on a knowledge test. In contrast to the findings of Hasler and colleagues , we founIn our experimental study, a schematic visualization was associated with fewer negative emotions and a more positive evaluation than a realistic visualization. This finding is particularly interesting, because recent technological developments have increasingly relied on schematic representations of surgical interventions . In addThe studies presented here have some limitations. First, the participants’ decision-making process was hypothetical, as our participants were not patients. Although this approach allowed us to perform the study in a controlled experimental setting and choose a situation that might be relatively easy to imagine, the motivation of our participants to engage in the situation may be lower than that among patients, for whom the situation would be personal and relevant. Patients may place importance on other aspects and react differently to the videos; in addition, some of our participants may have faced difficultly in imagining their feelings and thoughts in the described situation. Second, a large number of our participants were university students and therefore relatively young and well educated. Consequently, one should exercise caution when generalizing these findings to an entire population. For example, younger people may be physically more active and may therefore be more likely to opt for surgery than older people, which is supported by the fact that the average attitude toward surgery was positive. Some previous studies found that patients with low educational levels benefited significantly more from multimedia interventions than patients with high educational levels ,15,16. AWe informed participants that there were two possible treatment methods, but showed them a video that only addressed one method—surgery—because we wanted to focus on one intervention and clarify the reason for the possible effects observed. The disadvantage of this approach was that participants learned more about the surgery than about the alternative treatment, which likely influenced their attitude and decision-making process. Future studies with a larger sample size should create different videos about the different treatment options and compare their effects on the outcome variables. In addition, they should aim to transfer the research questions into more-realistic settings to determine if patients can benefit from such videos in the same way as the participants in our experiment did and show similar preferences regarding the video design.Medical educational videos are useful for providing knowledge to laypeople with little prior experience and support informed decision making. Our studies showed that the visualization format and user control options should be considered in the design of such videos. Our findings suggest that schematic videos with navigational options, along with encouragement to use them, may be most helpful in avoiding negative emotions and supporting knowledge acquisition.Videos are a good medium for educating patients about medical topics and should be used as decision-support tools to make complex information more vivid and easier to understand for laypeople. Our studies show that the design of such videos can influence information processing. The schematic visualization caused fewer negative emotions, was liked better than the realistic visualization, and was preferred by more than half of the participants in our survey. In contrast, almost one-fourth of the participants showed interest in a realistic presentation format. To increase satisfaction and personal benefit, different types of visualizations should be offered and patients should be given the opportunity to decide individually which type they prefer. This approach would be easy to realize in decision-support tools. Since participants spent more time with the video and acquired more knowledge with the use of navigational options, navigational options should be made available to participants and participants should be encouraged to actively use them, for instance, to pause and repeat difficult or interesting parts of the video.


Cancer patients often experience loss in body weight and also a decrease in muscle mass, which results in the reduction of physical activity and mobilization of the patient. To decelerate the loss of muscle mass, as part of the cancer treatment patients frequently undergo physical therapy and considering the physical capabilities of the patients, with moderate loads. Moreover, frequent studies also observed for cancer patients, together with the decrease in muscle mass a shift into fast-twitch muscle fibers from slow-twitch fibers. The aim of our study therefore was to determine how motor fibers behave under moderate isometric load executed until total exhaustion. 11 university students (G1), and 14 elite athletes (G2) participated in the study. 65% of the maximal voluntary contraction (MVC) was determined for the biceps brachii muscle, and with this load holding a weight, participants had to sustain a 90 deg. isometric elbow flexion in a standing posture until complete fatigue occurred. EMG activity for the biceps brachii muscle was measured and frequency analysis was performed. 3 windows were determined in the fatiguing protocol: the first (W1), middle (W2), and last (W3) 5 s, and also frequency analysis for MVC was performed (MAX) between 0 and 260 Hz with 20 Hz wide frequency bands. The results indicate, that as the protocol progressed in time and the effect of fatigue increased (from W1 to W3) the activity of low frequency muscle fibers significantly increased (0-40 Hz) while activity of high frequency muscle fibers (60-260 Hz) significantly decreased for G1 and G2 groups identically. We can conclude, that training applied with constant moderate tension as fatigue increases will result in the increased activation of the lower frequency slow–twitch muscle fibers, but the increase of fatigue in the lower frequency fibers will not result in the increase in the activation level of the higher frequency fast-twitch fibers. Consequently, because as slow-twitch fibers are being used at moderate loads and even when fatigue occurs in these fibers the fast-twitch fibers will not work, higher muscle loads are needed if the aim is to activate fast-twitch fibers. Considering the shift into fast-twitch muscle fibers from slow-twitch fibers for cancer patients, in some cases if the patient’s age and physical status allows during the physical treatment, higher loads and consequently higher levels of activation might be beneficial for the retardment of loss concerning the fast-twitch fiber mass. In cancer cachexia patients often face weight loss and also muscle atrophy, that results in the reduction of their quality of life, accelerates the progression of the cancer, results in worse surgical outcome and lower probability of survival –4.Although loss of muscle mass can often be observed in cancer patients, our present understanding of the phenomenon is restricted. Some studies conclude, that muscle atrophy is a result of cancer, but also cancer treatment can be associated with the loss of muscle mass , 6. To cConsequently, as in many cases for cancer patients, when high intensity training is not applicable, the question may arise as to whether exercises applied with moderate load will activate the fast-twitch muscles, and as a result reduce muscle mass loss in these fibers. When applying resistance training with moderate loads the slow-twitch fibers will be active, but it can be speculated, that if these slow-twitch fibers fatigue but the training continues, the fast-twitch fibers might take over the role and will become active.In the human neuromuscular system, the central nervous system regulates muscle force output by sending neural signals through alpha motoneurons to the muscles in two ways: by increasing the activation level if the signal, consequently recruiting and therefore activating more muscle fibers, and by altering the frequency of the stimuli . Low freThe aim of this study was to determine how the activation level of the biceps brahii muscle changes while the muscle reaches a completely fatigued state using moderate isometric load. To determine the activation level of different frequency muscle fibers, we have calculated the power spectrum content of the EMG data for 20 Hz wide frequency bands. The EMG signal, that can be recorded with surface electrodes, is a complex signal which consists of lower and higher frequency parts. Using the EMG software and Fourier analysis the lower and higher frequency components can be separated, and the amplitude, and consequently the intensity of the lower and higher frequency signals can be calculated, and as a result the activation level of the lower and higher frequency, slow and fast-twitch muscle fibers can be determined. Figure . Using m15 male and 10 female subjects participated in the study. The subjects were the students of the University of Physical Education. The subjects were in good physical condition, prior to the study they did not report any upper limb injury. Age: 23,83 ± 5,02 yrs., body mass: 70,71 ± 7,99 kg, height: 176,91 ± 7,13 cm. The sample was divided into two groups: recreational university students without sports background (G1) and professional handball players (G2) As the fiber characteristics for the two groups is different it can also be hypothesized, that the activation pattern of the frequency distribution for the two groups during the protocol would be different. As G1 group presumably has a mixed fiber type distribution, and G2 has a fast-twitch dominant fiber distribution, with this construction of the study we can replicate the phenomenon, that in the case of cancer patients with the decrease in muscle mass simultaneously a shift into fast-twitch muscle fibers from slow-twitch fibers occur [G1 consisted of 11 subjects , age: 21,63 ± 1,6 yrs., body mass: 69,45 ± 6,78 kg, height: 174,81 ± 5,09 cm, and G2 consisted of 14 subjects , age 25,57 ± 6,25 yrs., body mass 71,71 ± 10,74 kg, height 178,57 ± 9,33 cm, training age 13,71 ± 5,58 yrs. The subjects were informed about the study orally and signed a written agreement before participating in the study. The Ethical Committee of the University of Physical Education approved the protocol , which was in union with the Declaration of Helsinki.Surface EMG (sEMG) signals of the right biceps brachii muscle were recorded during the protocol. Before beginning the trial, the skin needed to be prepared to obtain better sEMG-recordings, to reduce the risk of imbalance between the electrodes, and noise. In order to achieve this, we removed the hair from the skin surface with a razor and the mortified epithelium with sandpaper, and finally washed the skin with alcohol . Two uniOur aim was to study muscle activation under submaximal isometric load with the use of a controlled external load. To achieve this first the subjects’ maximal voluntary contraction (MVC) of the elbow flexor muscles were measured in 90 degrees elbow flexion. The subjects had their arms flexed α = 90 degrees in supinated position, while standing on a Kistler Force Platform System 92–81 B and holding a fixed rod , middle 5 s (W2), and last 5 s (W3), and immediately after W3 signals for the maximal effort contraction was recorded (MAX), and also signals of the whole second MVC was processed. Between 0 and 260 Hz frequency spectrum intensity distribution was divided into 20 Hz frequency bands. Using the Noraxon software we have calculated the power spectrum content for the frequency analysis using Fourier transformation in the frequency bands.p < 0.05.The basic data was characterized by means and standard deviations. Because of the limited sample size and for the purpose of selecting the adequate statistical procedure Shapiro Wilk’s W test was calculated for checking the normal distribution of the data. The data was normally distributed, and for comparing the differences in different times for the same samples dependent T-test was used, for comparing the differences between the groups independent T-test was used. StatSoft STATISTICA 13.2 was used for the statistical analysis, Significance level was set for When comparing the data between the recreational university students (G1) and the professional handball players (G2) group, the G1 group had significantly greater activity in the W1 of 0–20 Hz frequency band, and MAX of 60–80 Hz frequency band, but we have not found significant difference for any other comparisons. As minimal difference could be observed in the EMG characteristics between the G1 and G2 groups, and taking into consideration the nature of EMG signals with relatively high standard deviations we have united the datasets for the two groups to calculate statistical difference between W1-W2-W3-Max to increase sample size (nG1 + G2 = 25 subjects), and consequently to increase the power of the statistical procedures and consequently the conclusions of the study. Comparing the results for the data of all of the participants significant increase  was found in 0–20 Hz frequency band between W1-W2 (inc. 162%), W1-W3 (inc. 618%), W2-W3 (inc. 174%); 20–40 Hz frequency band between W1-W2 (inc. 120%), W1-W3 (inc. 347%), W1-MAX (inc. 55%), W2-W3 (inc. 103%); 40–60 Hz frequency band between W1-W2 (inc. 78%), W1-W3 (inc. 28%); 60–80 Hz frequency band between W3-MAX (inc. 120%); 80–100 Hz frequency band between W3-MAX (inc. 163%); 100–120 Hz frequency band between W3-MAX (inc. 90%); significant decrease was found in 0–20 Hz frequency band between W2-MAX (dec. 50%), W3-MAX (dec. 82%); 20–40 Hz frequency band between W2-MAX (dec. 30%), W3-MAX (dec. 66%); 40–60 Hz frequency band between W2-W3 (dec. 28%), W2-MAX (dec. 30%); 60–80 Hz frequency band between W1-W3 (dec. 48%), W2-W3 (dec. 55%); 80–100 Hz frequency band between W1-W3 (dec. 70%), W2-W3 (dec. 63%); 100–120 Hz frequency band between W1-W3 (dec. 41%), W2-W3 (dec. 35%) ; 140–160 Hz frequency band between W3-MAX (inc. 143%); 160–180 Hz frequency band between W3-MAX (inc. 183%); 180–200 Hz frequency band between W2-MAX (inc. 65%), W3-MAX (inc. 176%); 200–220 Hz frequency band between W3-MAX (inc. 237%); 220–240 Hz frequency band between W3-MAX (inc. 195%); 240–260 Hz frequency band between W2-MAX (inc. 81%), W3-MAX (inc. 304%); significant decrease was found in 120–140 Hz frequency band between W1-W3 (dec. 57%), W2-W3 (dec. 53%); 140–160 Hz frequency band between W1-W3 (dec. 59%), W2-W3 (dec. 58%); 160–180 Hz frequency band between W1-W2 (dec. 36%), W1-W3 (dec. 72%), W2-W3 (dec. 56%); 180–200 Hz frequency band between W1-W2 (dec. 34%), W1-W3 (dec. 61%), W2-W3 (dec. 41%); 200–220 Hz frequency band between W1-W3 (dec. 61%), W2-W3 (dec. 52%); 220–240 Hz frequency band between W1-W2 (dec. 35%), W1-W3 (dec. 70%), W2-W3 (dec. 54%); 240–260 Hz frequency band between W1-W2 (dec. 52%), W1-W3 (dec. 69%), W2-W3 (dec. 55%)  of the individual is not a prohibiting factor, higher training load is beneficial for the retardment of loss in the fast-twitch fiber mass.


The current study examined whether problem drinking in older adulthood is associated with greater longitudinal risk of functional impairment and mortality through 2016. Problem drinking consists of patterns of alcohol use resulting in symptoms of alcohol dependence or health and social consequences. Participants were adults  from Wave 2  and Wave 3  of the Survey of Midlife Development in the United States (MIDUS) Study. Participants reported problem drinking behaviors  and any disability in basic and instrumental activities of daily living . Mortality data was acquired from the 2016 MIDUS Mortality dataset. Results indicated that 20.7% of the sample reported at least one problem drinking behavior in the past year. Multiple linear regression analyses revealed that the sum of problematic drinking behaviors at Wave 2 predicted 10-year longitudinal change in impairments in ADL’s  and iADL’s  after controlling for age, education, gender, and previous ADL/iADL. Logistic regression results revealed that for every additional alcohol use problem reported at MIDUS 2, the odds of mortality increased by 1.74 , beyond controls for age and number of chronic conditions. The findings that problem drinking has a unique positive association with impaired functioning and mortality risk during older adulthood supports public health efforts to encourage reduced consumption, increased medical screening, and expanded treatment options.


A near-fall is defined as a loss of balance that would result in a fall if sufficient balance recovery manoeuvres are not executed. Compared to falls, near-falls and its associated balance recovery manoeuvres have been understudied. Older adults may not recognise a near-fall or identify the use of their balance recovery manoeuvres to prevent a fall. The consensus on the methods to collect near-fall data is lacking. The primary objective of this study was to determine the feasibility of recruitment and retention. Secondary objectives were to establish evidence that Singapore community-dwelling older adults can identify near-falls and associated balance recovery manoeuvres. Texting and calling methods were explored as reporting methods.This study took place in Singapore (September to October 2019). Participants were healthy, community-dwelling adults aged 65 or older. Recruitment was done through poster advertisement, and all participants gave informed consent. Participants attended a briefing session and reported their near-fall or fall incidence over 21 days using either daily texting or calling. The primary outcome measures were the recruitment rate, retention rate, preferred modes for data reporting and ability to report near-falls or falls. Secondary outcomes included the self-reported incidence of falls and near-falls.Thirty older adults were recruited in 5 weeks. All participants completed the study. They understood near-fall concepts and were able to report the occurrence and relevant balance recovery manoeuvres used to prevent a fall. 87% (26/30) chose to text while 13% (4/30) selected calling as their reporting method. One actual fall (0.16%) out of 630 responses was reported. Thirty-six incidents (5.7%) of near-falls were recorded. Sixteen participants (53.3%) experienced near-falls and half of this group experienced two or more near-falls. The use of reach-to-grasp strategy (36%), compensatory stepping (52.8%), and other body regions (11.2%) were used to prevent the fall.The study provided evidence that studying near-falls in Singapore community-dwelling older adults is feasible and can be applied to a large-scale study. Recruitment and retention rates were good. Older adults were able to identify near-falls and balance recovery manoeuvres. Both texting and calling were feasible reporting methods, but texting was preferred.NCT04087551. Registered on September 12, 2019ClinicalTrials identifier: The online version contains supplementary material available at 10.1186/s40814-020-00748-1. What uncertainties existed regarding the feasibility?It is unknown whether Singapore community-dwelling older adults could determine near-falls and associated balance recovery manoeuvres.There was a lack of an operational definition of near-fall to be used in near-fall studies.There is no consensus on the reporting methods to collect near-fall data.2)What are the key feasibility findings?Singapore community-dwelling older adults were able to identify near-falls, and various balance recovery manoeuvres used to prevent falls such as compensatory stepping and reach-to-grasp strategies.The operational definition of near-fall and the reporting methods used in the study were well adopted by the Singapore community-dwelling older adults.There were good recruitment and retention of Singapore community-dwelling older adults in the study to investigate near-falls.3)What are the implications of the feasibility findings for the design of the main study?Singapore community-dwelling older adults were able to relate to the near-fall concepts, supporting that a large-scale study can be conducted to study near-falls.The feasibility of the operational definition of near-falls and data collection methods provided evidence to justify methods in the main study.The findings on the recruitment rate and retention rate may be used to justify a reasonable sample size to be calculated for a large-scale study.Falls in older people can lead to devastating health and social consequences, including serious injuries, hospitalisation, loss of independence, diminished quality of life and depression , 2. GlobNear-falls in older people are common and can be a risk factor for falls. Near-falls occur more frequently compared to an actual fall . More thWhile everyone tacitly knows what a near-fall is, a concrete definition of a near-fall has been lacking. A vague understanding can create ‘faulty transmission of information between patient and physician and between researchers.’ . HoweverVarious documentation methods to study falls such as questionnaires, fall diaries or telephone calls had been used widely . HoweverSingapore, being one of the most densely populated countries in the world , faces aTo evaluate the recruitment process and retention of community-dwelling older adults in a local study of near-falls.To assess the use of a briefing to explain the different definitions between a near-fall and a fall to the community-dwelling older adults.To determine the use of daily texting compared to calling as suitable data collection methods for an appropriate trial design relating to (i) participant preference (ii) adherence to protocol.This feasibility study was an observational cohort study. Data protection and ethical approval were obtained from two institutional ethics review boards, Singapore Institute of Technology (reference number: 2019129) and Queen Margaret University (reference number: REP0197). This study conforms with the CONSORT extension to randomised pilot and feasibility trials, excluding specific items required for randomisation nature of the study  (AdditioPoster advertisement was circulated to the network of older adults engaged by Singapore Institute of Technology (SIT) for school assignments, residents’ network centres and various clinical partners across Singapore. Between September and October 2019, interested older adults contacted the researcher through the contact details listed in the posters or given through word-of-mouth recommendations. They were asked by a researcher whether they were aged 65 years or older, living in the community and were able to read, write and communicate in English before a meeting was arranged at SIT or an agreed location in Singapore. During the meeting, they were provided study information, e.g. how the study would be conducted, what will be expected of them, the study’s eligibility criteria , cognitive functioning using the Six-item Cognitive Impairment Test (6CIT) , upper lThe 6CIT is a brief and simple validated tool used for cognitive screening in the community-dwelling older adults . ParticiThe HRT  is a perThe TUG is a reliable and valid test for quantifying functional mobility in older adults . ParticiOne primary outcome measure was to determine the feasibility of conducting a presentation to explain the different meanings between a near-fall and a fall to the community-dwelling older adults. Operational definitions of falls and near-falls were presented to the participants. These definitions were consistent to those in the literature using language and concepts that aimed to be clear, relevant and easily understandable by the older participants.A fall definition was adopted, in concordance with the PROFANE-group consensus statement, as ‘an unexpected event in which the participant comes to rest on the ground, floor or lower-level’ . ExplainNear-fall was defined as an event when the individual slips, trips or loses balance but uses the hand(s) or leg(s) or any body part to recover balance and prevent a complete fall. This definition aimed to be relevant, comprehensive and understandable to the older participants. Participants were then presented with several scenarios Table  and askeAfter the briefing, the researcher obtained the preference of the participants’ mode of communication to report the incidence of near-fall, fall or no fall. Three options were provided: (1) a daily call or (2) a daily text or (3) either a daily call or text scheduled at a prearranged timing. There were no text reminders given during the day to avoid overburdening the participants. One scheduled text was sent each day, even if there was no text reply from the participant. For calling, a second call would be made an hour later of the scheduled timing if there was no response to the first call. No further calls were made if there was no response to the second call. Over the next 21 days, the participants were asked two questions by the researcher daily, ‘Have you fallen in the past 24 hours?’ and ‘Have you almost fallen in the past 24 hours?’ using the participant’s preferred mode of communication. If ‘yes’ was replied to the near-fall question, the participants were asked, ‘Did you prevent the fall using your hands or legs or any body part?’ The participants then identified the balance recovery manoeuvre used to prevent the fall. If ‘yes’ was replied to the fall question, the researcher checked if the participant was able to continue with the study. A 21-day follow-up duration was selected to replicate the study period applied by Ryan and colleagues . All datThe feasibility outcomes were summarized descriptively and narratively. Descriptive statistics were used to summarize recruitment, retention, sample characteristics, incidence frequency of near-falls and falls and the types of balance recovery mechanisms used in near-falls.n = 14) were (a) 2 adults did not meet age criteria and (b) 12 older adults were non-English speaking, e.g. Mandarin. The remaining 30 participants met the eligibility criteria and were enrolled in the study. See Fig. The recruitment for the study was estimated to be at a rate of three to five participants per week, and the study was projected to complete in 8–12 weeks. The study completed within 8 weeks because of the outreach to the network of older adults formerly recruited for the school’s projects and the strong links that the research team has with the various community groups in Singapore. Of the 44 community-dwelling older adults screened, 30 were eligible for this study. The reasons for the exclusion  compared to female , aged between 65 and 85 years (72 ± 5.2). There was a fair ethnic representation of the Singaporean community with Chinese , Malay , Indian  and Eurasian . There was mixed participation of individuals with different education level, including primary level (10%), secondary level (53%) and tertiary level (37%). About half of the group lived in a 5-room or executive flat-type property and the others in 3-room flat (16.7%), 4-room flat (13.3%) and private housing estates (23.4%). Most of them were living with others (90%). All were community ambulant with some using a walking stick in the community (10%). Thirteen participants (43.3%) had fallen at least once in the last year, and 10 participants (33.3%) had experienced one or more near-falls in the last month. Nineteen participants (63.3%) reported they have never or rarely experienced a near-fall in the last one year while 11 (36.7%) reported occasional or frequent near-falls.The participants’ characteristics were presented in Table Out of the thirty participants, nineteen requested texting, two participants chose to have calling, and the remaining nine had no preference to either texting or calling. For those with no preference, seven participants eventually chose texting because they wanted the convenience to report their data at their availability. Two participants decided to receive a call because they preferred to talk to the researcher and wanted to address any concerns that might arise during the study period. During the study, one participant switched from calling to texting because she reported having difficulty talking due to her gum pain.Compared to texting, calling was preferred when clarifications were needed. Two participants who chose to text, called the researcher to clarify their near-fall. One participant asked if the event was a near-fall when he almost fell after he unintentionally bumped against another pedestrian while he was walking and using his phone. Another described his experience that he recovered using his body without the use of the hands and legs after losing balance on a chair. The researcher recorded these incidents as near-falls, and the balance recovery manoeuvres were the use of the hand and other body parts, respectively. The study did not record the details of these events. It may be beneficial for future studies should investigate the older peoples’ near-fall experiences.All participants completed a 30-min one-to-one briefing session using a PowerPoint presentation. The use of short video clips illustrated different near-fall scenarios to aid participants’ understanding of the concept of near-falls. All participants reported no issues in identifying and differentiating between falls and near-falls. All correctly answered the six test scenarios.All participants reported their near-fall and fall incidence during the study period. A total of 630 events  (100%) were recorded across the study period. The data received by the researcher was not reported daily by some participants. Over the 21-day period, five participants text-replied on the second day and one participant text-replied on the fourth day in one occasion. One participant text-replied on the second day across four episodes and one participant varied his text-replies up to 4 days through the study. For calling, all participants needed more than one scheduled daily calls to obtain the data over the study period.None of the participants reported any difficulty remembering whether they had a fall or near-fall during the study period. The concepts of falls and near-falls were well-understood by all participants. However, clarifications were needed for the ‘other body parts’ balance recovery manoeuvres used to recover from a loss of balance, i.e. ‘using the hip to lean against the wall’ or ‘body jerking up’.During the 3 weeks, one actual fall was recorded. This yielded a fall incidence of 0.1% (1 in 630 records). Near-falls were reported 36 times or an incidence rate of 5.7%. Among the thirty participants, sixteen participants (53.3%) experienced near-falls, and 50% of them experienced two or more near-falls. A comparison was made between older adults who experienced one or more near-fall  and those who did not experience a near-fall  during the study Table . The neaThis study had good participation and retention rate. There was an average of five older people recruited into the study each week, completing the recruitment in 5 weeks. There was no difficulty recruiting older people who were able to communicate in English. The profile of the sample suitably represented the Singapore older people community as a multi-ethnic society. They were able to report their encounters with near-falls (if any) based on their interactions between their regular activities of daily living and the environment. However, we found that some older participants were unable to participate in this study as the ability to communicate English was listed as an eligibility criterion. For a future larger-scale study, the study materials may be translated into different languages, such as Mandarin, Malay and Tamil to allow greater participation of older people. In regard to retention, the high adherence of participants in our study was consistent with the study conducted by Ryan and colleagues . It is pA 30-minute briefing session conducted was sufficient to ensure that adequate information of the study provided enough understanding to older people, without causing unnecessary mental fatigue . While tThe clarifications requested by two participants about near-falls implied some improvement would be needed for the operational definition of near-falls. In the study, the definition of a near-fall was presented as an event where the individual slips, trips or loses balance but uses the hand(s), (leg)s or any body parts to recovery balance and prevent a complete fall. In future, the different types of events (i.e. the type of perturbations) and various balance recovery manoeuvres (i.e. use of the hand(s), leg(s) or body parts) could be further elaborated with the various causes of disequilibrium and the different ways to recover equilibrium respectively. One suggestion would be to define a near-fall as an event when the individual may experience a fall due to external perturbations, such as a slip, a trip or external forces causing the individual to lose balance, or due to internal perturbations such as the movement of the individual resulting in the individual being destabilised . The othData collection methods, such as telephone calls and diary, had been used in previous studies to record near-falls , 20. TheThe evidence in this feasibility study demonstrated a positive predisposition towards the use of texting as a choice of data reporting in Singapore. Both methods using texting and calling were highly successful for recording near-falls or falls. Both participants and the study team appreciated the flexibility of using texting. While scheduled timings were arranged with the participants, not all participants replied immediately to the text messages or picked up the call. All participants reported their data at their own time. It was noted that calling is a more time-consuming method on the part of the researcher to reach the participant. To reduce overburdening of the researchers, a larger-scale study would need to factor in the necessary resources  to conduct the calling.Nevertheless, both ways confirmed these methods’ potential use for obtaining data that may be easily forgettable. Based on participants’ feedback and the researchers’ subjective impressions, the frequency using a twice-a-week interval to get falls or near-falls data is practical to implement, able to sustain the cooperation of the participants and easy for participants to retain any fall or near-fall details.This study demonstrated that the procedures and data collection processes were feasible. It provided evidence that older people can understand the concept of near-falls and identify relevant balance recovery manoeuvres used to prevent a fall. The working definition for falls and near-falls described in this study is comprehensible and relevant to the older adults. Both texting and calling are feasible reporting methods, but older adults in Singapore preferred texting as the more convenient mode of reporting.Additional file 1: Table S5. CONSORT extension for Pilot and Feasibility Trials Checklist. CONSORT 2010 checklist of information to include when reporting a pilot or feasibility trial. A completed CONSORT extension for Pilot and Feasibility Trials checklist.


A DVI operation for an incident characterised by many fragmented and otherwise compromised human remains poses specific challenges that may prolong and complicate identifications.Specialists should be consulted at the outset to address key issues related to the aim and extent of the operation.Specialist expertise in handling compromised human remains is indispensable at the scene, in the mortuary, during reconciliation, and for quality control.Continuous consultation between representatives from government, the judiciary, law enforcement, the media, and various forensic specialists will prevent unnecessary delay and facilitate accurate and timely communication.Human-made and natural disasters can result in severely fragmented, compromised, and commingled human remains. The related disaster victim identification (DVI) operations are invariably challenging, with the state of the remains potentially precluding some identifications. Practitioners involved in these DVI operations will routinely face logistical, practical, and ethical challenges. This review provides information and guidance derived from first-hand experiences to individuals tasked with managing DVI operations with fragmented human remains. We outline several key issues that should be addressed during disaster preparedness planning and at the outset of an operation, when incident-specific strategies are developed. Specific challenges during recovery and examination of fragmented remains are addressed, highlighting the importance of experienced specialists at the scene and in the mortuary. DNA sample selection and sampling techniques are reviewed, as well as downstream effects of commingling and contamination, which can complicate reconciliation and emphasise the need for rigorous quality control. We also touch on issues that may arise during communication with families. While recommendations are provided, they are not intended as proscriptive policy but rather as an addition to the general recommendations given in the International Criminal Police Organization (INTERPOL) DVI Guide, to inform preparative discussions between government officials, judiciary, police, and forensic specialists. Disasters, either natural or human-made, are inherently chaotic events with major adverse effects on infrastructure, environment, and communities. When a disaster involves large-scale loss of life, the recovery process is further complicated by a simultaneous identification operation. The primary aim of a disaster victim identification (DVI) operation is to recover all human remains, identify the deceased, and certify their cause and manner of death. Additionally, the investigations related to DVI may be instrumental in reconstructing the cause of the event, which can facilitate the development of preventive measures.In 1984, the international police network International Criminal Police Organization (INTERPOL) introduced the first written DVI guidelines. The INTERPOL DVI Guide is published and regularly updated by a DVI working group advised by four scientific sub-working groups related to the areas of forensic expertise generally consulted in DVI operations: odontology, pathology/anthropology, ridgeology (fingerprints), and molecular biology (DNA). DVI operations worldwide typically follow the INTERPOL DVI Guide and use its accompanying documentation forms . The docIncidents with severely fragmented, compromised, or commingled human remains pose complex logistical, practical, and ethical challenges that may prolong the DVI operation or even preclude identification of some individuals altogether. Maximising identification efforts in such circumstances requires a different approach to documentation in the mortuary, as well as detailed planning and effective communication between government officials and forensic practitioners. The importance of an augmented approach to complex incidents is even greater with the increased sensitivity of DNA techniques and more sophisticated ways to interpret mixed or partial DNA profiles. These developments have made it possible to identify even the smallest piece of human tissue ,4, but tFurthermore, there are limited experience-based resources for addressing the abovementioned challenges. This article therefore presents an overview of lessons learned from several complex DVI operations, all characterised by severely fragmented and compromised human remains. First, complex operations in which the authors have been involved are summarised, followed by a discussion of key issues in DVI preparedness planning. Remains recovery and examination, DNA sampling, reconciliation, and quality control are reviewed, as is a consideration of issues that may arise during communication with community groups and next of kin.The recommendations listed in this publication are based on the first-hand experiences of the authors, all of whom are experienced forensic anthropologists with one (HdB) also being a forensic pathologist. The authors have worked on numerous DVI operations with many fragmented remains; a summary of the major incidents that form the basis of the recommendations is provided below.2, was 43 m deep, and had three “hot spots” burning for almost 3 months. The fires were periodically doused with brackish water from the nearby East River. It took nearly 9 months to comb the debris field and recover approximately 22 000 fragments of victim remains. These human remains were extremely fragmentary, decomposed, burnt, and commingled. More than 3 years were required to finalise the missing persons list. As of August 2019, 14 696 human fragments have been identified and matched to 1 637 of the 2 749 victims. Primary mortuary operations lasted approximately 11 months and were undertaken by a multidisciplinary team of medical examiners, an anthropologist, New York Police Department fingerprint examiners, forensic odontologists, X-ray technicians, medicolegal investigators, DNA analysts, and hundreds of volunteers. Quality control checks and reanalysis lasted an additional 6 months after initial analyses were complete in the mortuary, and DNA testing and resampling are still ongoing.On 11 September 2001, nine terrorists flew passenger aircraft American Airlines 11 and United Airlines 175 into the north and south towers of the World Trade Center in New York City. Within 2 h, both towers collapsed, killing nearly all civilians, first responders, and emergency personnel inside. The disaster site covered 64 500 mOn 12 November 2001, passenger aircraft American Airlines 587 crashed in a residential neighbourhood in Queens, New York less than 2 min after take-off. All 260 people on board, along with five individuals on the ground, were killed. The National Transportation Safety Board determined the cause of the crash to be a combination of an airline design flaw and pilot error . The rem2 that resulted in the deaths of 173 people. Total extinguishment of the fires took approximately 6 weeks [On 7 February 2009, the state of Victoria, Australia experienced extreme heat and strong winds. In combination with faulty overhead power lines, arsonists, and lightning strikes, this heat event caused a total of over 300 bushfires in 4 500 km 6 weeks . Very fe2 crash site submitted for examination.On 17 July 2014, passenger flight Malaysia Airlines 17 from Amsterdam to Kuala Lumpur was shot down while flying over Ukraine, killing all 283 passengers and 15 crew members on board . An armeOn 22 August 2015, 11 people were killed when a vintage Hawker Hunter jet crashed onto a busy dual carriageway during an airshow in West Sussex, UK. The impact from the jet caused major bodily disruption, with dispersal and commingling of body parts across a wide area. The search and recovery of the remains took approximately 3 weeks, with further damage and decomposition of human remains caused by fluctuating weather conditions. Despite the relatively small number of victims, more than 1 200 body parts were recovered and the examinations lasted approximately 6 weeks, with numerous re-examinations required because of commingling and potential cross-contamination. All individuals were identified. A forensic anthropologist and archaeologist assisted at the scene. A forensic pathologist led the mortuary examinations during the first 5 d, focussing on the cause of death and larger body parts. An anthropologist assumed responsibility for the subsequent 5 weeks of examinations, assisted by anatomical pathology technicians and a police DVI mortuary team.On the evening of 13 November 2015, three groups of men launched six separate assaults—suicide bombings and mass shootings—in the centre of Paris and the northern suburb of Saint-Denis. In total, 130 people were killed . Many inOn 5 February 2019, around midnight, a fire in a Paris apartment block killed 10 people and injured an additional 40. The fire was alleged to have been started intentionally. The fire was extremely intense, quickly consuming several floors. Eighty-five fragmented and charred bodies/body parts were recovered under supervision of a forensic anthropologist and odontologist. The charred bodies and body parts were first CT-scanned, after which radiologists and the anthropologist triaged the remains. The remains were subsequently examined by a multidisciplinary team of forensic pathologists, odontologists, and a forensic anthropologist in close collaboration with a DVI police unit. All individuals were identified over 4 days. Most of the victims were identified using DNA.Each disaster is unique, and the final decision on the mandate, extent, and procedure of each DVI operation is the prerogative of the local legal authorities. As such, the lessons learned set out below are not intended as proscriptive policy. Rather, they are meant to guide preparative discussions between relevant government officials, judiciary, police, and forensic specialists. The discussion is not meant to supplant the INTERPOL DVI Guide, but to provide additional information specifically for incidents with many fragmented remains.Who will advise the responsible decision-makers? In the moments following an incident, obtaining reliable information is key to the timeliness of remains recovery and establishing identifications. The importance of providing experienced and specialist advice to the local authorities at the scene of the disaster cannot be overstated, especially when dealing with compromised bodies.Who will be responsible for the DVI operation, and what is the chain of command? Typically, local authorities will have addressed this in their response plan. However, confusion about the role of forensic specialists may still cause considerable delay, particularly if there are multiple scenes.Which experts are required at the scene? Relevant expertise at the scene, especially forensic archaeologists or anthropologists when human remains are fragmented or burnt, has proven to be essential for efficient recovery. The scale of the 2009 Victorian bushfire incident, coupled with multiple concurrent scenes and limited resources, highlights the need for expertise at the scene. Many of the initial scene attendances did not include a forensic anthropologist or archaeologist, experienced with recovering small burnt and fragmentary bones, and therefore required numerous site revisits. The same was true following the World Trade Center recovery operation, which was predominantly undertaken by the New York Fire Department. Their inexperience resulted in avoidable and additional commingling at the site, which then had to be addressed during the mortuary process. The number of deceased is not the single most important factor when selecting specialists; at the Rue Erlanger fire incident in Paris, an anthropologist and odontologist attended the scene for (only) 10 fatalities to aid in recovery and prevent additional commingling.Forensic anthropologists routinely attend fatal fires in the UK to record the location of victims and disassociated fragments, which can be complex. For example, in 2017, six family members were killed in a fire in Wales. The victims had fallen through several floors and some were commingled, giving the impression that they had been together at the time of death. Careful excavation and examination of the remains by forensic anthropologists and archaeologists established that this was not the case and that they had, in fact, been in separate rooms in different parts of the house. Scene analysis contributed to the interpretation of what had occurred on the night of the fire, and proved crucial to the coronial and police investigations. These examples highlight the fact that even when the number of victims is relatively low, specialist assistance at the scene can significantly contribute to a successful DVI operation, particularly if the remains are extremely fragmented and dispersed or exhibit significant thermal alterations and commingled.To what extent will postmortem examinations be undertaken? This decision is often context-specific. When the cause of death is assumed, or there is no suspicion of criminal intent, local authorities may decide that the examination of the remains will be limited to identification. This can minimise the mortuary phase of the DVI operation considerably. For example, cause and manner of death from the World Trade Center disaster were predominantly homicide/blunt force trauma, and autopsies were not conducted. However, during the DVI operation for the victims of American Airlines 587, autopsies were legally mandated by federal laws. During the Malaysian Airlines 17 operation, autopsies were limited to specific persons of interest, such as the pilots. Close communication with the local authorities is vital. Following the 2009 Victorian bushfires, any suspicion of a death other than by fire was communicated to the coroner and the extent of the postmortem examination was reviewed .What is the identification mandate? Will every fragment be identified, if possible, or will the sole goal be victim identification? In disasters with a closed population and a known missing persons list, authorities may decide to end the DVI operation as soon as each individual is accounted for. In an open-population disaster, an unknown number of victims precludes such an approach. Discussion should then focus on the level of identification, i.e., what type or size of human remains is considered “identifiable”. Often, this decision is dictated by resource availability.Terminology must be clear and specific. Non-specific terminology may cause confusion or even lead to erroneous conclusions. For example, terms such as “full body”, “body part”, and “fragment” should be clearly defined. Specific descriptions are also needed to clarify context when interdisciplinary personnel are working together on a DVI operation. For example, the debriefing following the 15 March 2019 mass shootings at mosques in Christchurch, New Zealand revealed that to DVI forensic medical experts, the term “fragmentation” indicated body preservation, whereas for police investigators, the term related to ballistic evidence .How will information be conveyed to families of the victims, the broader community, and government officials? Families may have specific questions regarding potential survival time and completeness of the body. Managing expectations and communication with next of kin are imperative at the outset, especially if it is possible that a number of fragments would not be identified. Additionally, special attention should be paid to how body fragmentation may impact specific religious and cultural preferences. For example, some religions consider it essential to recover all body parts for burial. Realistic expectations should be communicated to all relevant parties simultaneously and consistently.Is there a media communication strategy? Well-informed and timely media briefings ensure that misinformation is not disseminated. A dedicated spokesperson will contribute to uniformity in the shared information.All abovementioned incidents have shown that a dedicated strategy to managing human remains will help ensure relative order in the complex situation following mass fatality. Ideally, this strategy should be part of the disaster preparedness plan rather than reactionary. Because each disaster carries its own intricacies, such a plan should not be restrictive and should include an outline of key topics that should be addressed at the outset of a DVI process. Ideally, these topics should include the following considerations:Typically, the complexity and duration of a DVI operation with many fragmented remains are underestimated. Discussion of the abovementioned topics with all stakeholders can mitigate this to some extent. The need for rigorous, quality-checked identification methods should always prevail over speedy identifications.in situ.Incorrect recovery techniques at the scene adversely affect mortuary operations, as demonstrated in some of the DVI operations above. Examples include grouping multiple sets of fragmentary remains in the same recovery bag and attempted body reconstruction at the scene . To circNo two disaster sites are identical; therefore there is no single approach suitable for every context. For example, an unmanned aerial vehicle survey and laser scanning might be appropriate to map a large open area such as a plane crash site, where remains are scattered across the ground surface ,17. Howein situ, which allows re-assignations with larger, identifiable body parts in the mortuary. This approach was employed by a forensic anthropologist while excavating a tank that had been hit by an improvised explosive device in Afghanistan in 2012, killing all six soldiers on board. The large amount of ordnance that they had been carrying intensified the explosion and prolonged the burning inside the vehicle. Prior to excavation and recovery of the fragments, a plan of the vehicle’s interior was made, dividing it into zones corresponding with the original locations of the soldiers. This assisted greatly with the re-assignation of body parts and the preliminary identifications in Afghanistan. Formal identifications were completed in the UK following joint examinations with a forensic pathologist.Special consideration should be given when recovering burnt remains, as they are often highly fragmented and extremely fragile. They may be dispersed over a wide area, commingled with the remains of other individuals, or mixed in with other non-human material. Intense heat can alter the size, colour, shape, and mechanical properties of human tissue, making the recognition of burnt remains difficult . If boneQuality assurance should be integral to the recovery process. If the recovery is not undertaken by a specialist with expertise in compromised remains, the victim recovery forms accompanying the body parts should be reviewed before the body bags are sealed. This ensures that the descriptions of remains are correct and that accurate sketch plans of locations have been completed. A new form for recording and packaging highly fragmented human remains has recently been designed  and is cForensic anthropologists are often the specialists that assist with compromised human remains , 23–26. Full bodies and larger body parts are typically prioritised at the beginning of a DVI operation, with fragments addressed later in the process. Forensic practitioners, however, have an ethical responsibility to examine all human remains regardless of size or preservation status and despite their specific challenges . Small fWhile the initial examination involves triage, a more detailed examination, conducted as part of the mortuary assembly line, is geared toward identification. There is significant value in this examination being undertaken by a forensic anthropologist while a forensic pathologist attends to the larger, more readily identifiable bodies or body parts. Simultaneous examination ensures that the fragments are sampled for DNA as soon as possible, instead of waiting until the larger parts have been processed. This not only limits further decomposition and degradation but also allows for identified bodies or body parts to serve as DNA exemplars for unidentifiable fragments without identifying characteristics. At the same time, in instances with closed populations and limited resources, the possibility of 100% victim identification may be achieved before 100% fragment identification.A stepwise approach is best for the detailed examination of fragments, initially removing non-human material inadvertently collected at the scene and then separating duplicate skeletal elements, an indicator of commingling. Re-association of fragments may reduce the number of DNA samples needed. This should, however, be limited to actual physical articulation of fractured bones, and the slightest suspicion of commingling should prompt separation of fragments. As fragmented remains may be separated and re-associated with other fragments several times during the DVI operation, a rigorous documentation strategy that includes photographs and chain of custody is required. This will prove extremely valuable should future re-examination and re-sampling become necessary.A detailed examination of each fragment should always be performed, initially to determine the presence of a primary identifier outside DNA, i.e., fingerprints or odontological information. Additionally, it is necessary to record specific features that may assist with positive identification  or the reconstruction of the events (such as relevant forensic findings or patterns of perimortem trauma) . HoweverIncidents with prolonged recovery operations and thousands of fragments may require weeks, months, or even years to complete the analysis of all remains. During this period, the remains should be stored in such a way that minimises further decomposition. One option is to keep the remains frozen, preferably at −80 °C, although the necessary infrastructure may be prohibitive for larger fragments. An alternative approach is to desiccate the remains prior to storage. DNA testing following desiccation of remains from the World Trade Center disaster indicates that this method prevents further decomposition without negatively impacting genomic information.Numerous studies , 34–40 hVarious studies ,35,38,41Bone outperformed tooth samples in the Malaysia Airlines 17 investigation . This fiDuring the DVI operation, close liaison between the mortuary and the DNA laboratory increases operational efficiency . DNA speWith fewer practical limitations on DNA testing, the decision whether to test every fragment becomes an important consideration early in the operation. Authorities should be fully cognizant of the effects of a decision to do “everything possible”. Not only can this lead to potentially unrealistic expectations , but it will also increase the cost and duration of the DVI operation considerably. Moreover, it will significantly impact the regional availability of DNA analysis outside the DVI operation.Sampling DNA in a DVI mortuary does not differ much from sampling DNA during normal autopsy practice. However, special attention should be paid to limit cross-contamination. In soft tissue samples, this means that after any incision through an outer layer, the scalpel should be discarded (if disposable) or decontaminated with 10% bleach prior to obtaining an uncontaminated sample from the inner tissue. Bone can be sampled using a disposable scalpel if it is an intact specimen such as patella or foot bone. Sampling intact bones can also prevent additional contamination during bone sectioning. Additionally, bone can be sampled by hand-sawing a section using a junior hacksaw or autopsy saw, although this may require electricity and can be labour-intensive, particularly if practitioners are sampling hundreds of cases per day. Unless disposable, the blade should be cleaned or changed between samples. A stepwise manual for sampling soft tissue, bone, and teeth using only readily available, inexpensive instruments is provided in de Boer et al. . PreferaAs outlined in the INTERPOL DVI Guide, standards for identification should be based on reconciliation of antemortem and postmortem data from at least one primary identifier. In the case of fragmented remains, most matches will be based on DNA, and only a minority of fragments will provide another (corroborating) primary identifier such as odontology or fingerprints. While most jurisdictions will accept a single primary identifier, it is good practise to corroborate such an identification with available secondary identifiers such as associated material items . Alternatively, if resources are limited, the DNA sample from a case identified by another primary modality can be stored frozen without being processed. If questions later arise about that specific sample or identification, the DNA sample will still be available to test. Furthermore, the DNA from one fragment identified using dental methods can be used as a direct exemplar to re-associate additional fragments, instead of seeking sufficient familial DNA to make indirect matches; this avoids delays in processing family reference samples and simplifies matching statistics.As an additional quality check, fragments identified using DNA should be compared with previously identified pieces from that particular individual. The best practice is to use a body diagram to illustrate which body parts were recovered and which are still absent . Using sIncidents with many fragments pose specific challenges when communicating with the families of the deceased. Although best practice dictates separating and identifying all pieces originating from a single individual, small commingled fragments or segments of decomposed soft tissue may remain unidentifiable at the end of the process. The families should be made aware of this for transparency and local authorities should plan how to handle this issue.Additionally, given the time-consuming process of recovering and identifying fragmented remains, it is likely that fragments from the same individual will be identified weeks, months, or even years apart. Consequently, families may have specific wishes as to how they should be kept informed. Implementing a process for next of kin to make these considerations early, with the freedom to change their minds at any time, can be facilitated by a form outlining several options. For example, some may want to be notified each time a new fragment is identified, while others may wish to be informed of only the first fragment or only at the end of the process. Experience shows that this is an individual decision and often changes as time from the incident extends. Furthermore, family members may express disparate views and wishes .Consideration should be given to how the extent of disruption and the completeness of the body would be communicated to family members. At times, family members may request various types of information and differing levels of detail. To facilitate communication, the DVI team can present various prearranged options, ranging from full file review, to a review without photos, to more removed versions of the data such as diagrams. Following the 2015 Shoreham air crash, discussions were held between the forensic anthropologist, the senior coroner, and the family liaison coordinator to consider various options, including virtual three-dimensional reconstruction of the body parts using postmortem CT data. This idea was ultimately rejected as it was considered too graphic. Instead, a body diagram was used to convey each individual’s completeness. A table that listed the identified body parts was also created, with one column using scientific terminology and the other non-scientific terms.Incidents in which a perpetrator’s remains are commingled with the victim’s remains pose unique challenges, as victims’ families often request that the perpetrator’s remains be separated from the victim’s. Depending on the condition of the remains and whether DNA or other forms of antemortem data are available for the perpetrators, this may or may not be possible. Communication on this should be clear and honest, to manage family expectations without promising unattainable goals.Another important consideration is the presentation of evidence in courts and at coroners’ inquests. The way in which evidence is presented has been shown to impact how juries and families understand the evidence ,47. DiscThe format of each DVI operation is dictated by the context of the event, including incident type, the number of victims, the condition of the remains, and the decisions made by local authorities, which typically include government officials such as emergency planners and councillors, coroners/public prosecutors, police, and other emergency services. As such, each DVI operation is unique, making it difficult to provide specific recommendations beyond the general ones presented in the INTERPOL DVI Guide. However, the combined experience of the authors illustrates similar specific challenges arising from disasters characterised by many fragmentary and compromised remains.Communication between local authorities and forensic specialists is pivotal to ensuring a timely and efficient identification effort. Pre-disaster planning is essential and discussions between local authorities and forensic specialists should occur as soon as possible following the disaster. Agreements should be reached concerning the role of relevant forensic specialists at the scene and in the mortuary, and a flexible plan should be formulated for quality assurance, recovery, examination, and repatriation of remains.


Scientific Reportshttps://doi.org/10.1038/s41598-020-66615-4, published online 18 June 2020Correction to: ‘Supplementary Information Video 1b’‘Supplementary Information Video 1c’‘Supplementary Information Video 1d’‘Supplementary Information Video 3’‘Supplementary Information Video 4’This has been corrected in the HTML version the article. The PDF was correct at the time of publishing.The following supplementary files were omitted from the original version of this Article: