The characteristics and state of knowledge of bioelectric signals such as ECG, EEG, and EMG are initially discussed. This serves as the basis for exploration of the degree of scholastic coverage and understanding of the level of clinical acceptance of respective bioelectric signal subtypes during the last 60 or so years. The review further proceeds to discuss surface EMG (sEMG). The status of the field in terms of teaching and academic training related to sEMG is examined, and its clinical acceptance in several areas of medicine and kinesiology, including neurology, psychology, psychiatry, physiatry, physical medicine and rehabilitation, biomechanics and motor control, and gnathology, is evaluated. A realistic overview of the clinical utility of the measurement of sEMG signals and their interpretation and usage, as well as of perspectives on its development, are then provided. The main focus is on the state of the field in Croatia. EMG signals are viewed as “windows” into the function of the neuro-muscular system, a complex and hierarchically organized system that controls human body posture and gross body movement. New technical and technological means to enable the detection and measurement of these signals will contribute to increased clinical acceptance, provided current scientific, educational, and financial obstacles can be removed. Since its beginnings around the mid-twentieth century, the field of surface electromyography (sEMG)  has evolHere, our aim is to critically reflect on the position of this measurement technique in both educational curricula  and the clinical environment. The starting point of this endeavor is a discussion of the relationship of sEMG with tools that rely on the acquisition and interpretation of other types of common bioelectrical signals. In particular, the status of the field in Croatia is considered.Changes in bioelectric potential originate in particular organs and organ systems. Because bodily tissues serve as an electrical conductor, potential changes generated spread through the body and reach the body surface, where they are amenable to detection by suitable technical means. In the following sections, the types of bioelectric signals most commonly recorded are succinctly depicted: an electrocardiogram (ECG) originates in cardiac muscle; an electroencephalogram (EEG) originates in the brain; and an electromyogram (EMG) originates in skeletal muscle(s).The concept of membrane potential is central to bioelectric signal generation and conduction. The process of generation of change of equilibrium potential takes place at the cell membrane, which in a resting state maintains an electrical potential in the range of −40−90 mV , the state of equilibrium being described by the Nernst equation , 3. WhenThe contracting heart muscle generates electrical potentials. Numerous studies have modeled the mechanical action of heart muscle and performed bioelectric imaging . Clark pBecause cardiac contractions are a repetitive and continuous process, the ECG exhibits a characteristic quasi-periodic waveform. Since the beginnings of the analysis of the bioelectric characteristics of the heart muscle, the ECG technique has become established as a reliable, important, and practical diagnostic tool. Clinical standards have since been developed for the diagnosis of arrhythmias, ischemias, and numerous other pathological conditions.Beginning in the 1960s, computer-aided analysis of ECG was introduced , and, ovSince Hans Berger, a German psychiatrist, systematically analyzed the electrical activity of the brain for the first time, the EEG technique has gained prominence in neurology, contributing significantly to neurological diagnostics, and additionally proving useful in the fields of neuropsychiatry and psychology . The fluTypical clinical EEG waveforms recorded using scalp electrodes  can be identified. Correlations exist with specific brain states  and specific pathologies associated with abnormal EEG waveforms. EEG frequency ranges are as follows: delta [below 3.5 Hz ], theta (4–7.5 Hz), alpha (8–13 Hz), and beta  .Computer-aided EEG analysis has mainly been used for monitoring sleep and certain pathologic states, leaving the more difficult problem of diagnosis to the expert neurologist . AnotherThe field of electromyography encompasses both surface and intramuscular EMG: here, however we focus exclusively on sEMG. Although sEMG provides a global view of skeletal muscle function, in principle, the analysis of multielectrode recordings enables assessment of the activity of individual motor units (MU) as well . In addiMuscular contraction is a mechanical event involving the transformation of metabolic energy into mechanical force and power. Leaving the mechanical aspects of muscular contraction aside , here weThe total bioelectric signal of a muscle is the result of spatiotemporal summation of the activity of a large number of MUs, producing what is referred to as an interference pattern. Basmajian and De Luca presented a mathematical model of the myoelectric signal , 12, 13.Individual fiber potentials—recording of which would pre-suppose needle detection—sum up to represent a motor unit action potential (MUAP). A wide range of neuromuscular disorders alter the MUAP waveform in different but characteristic combinations, the interpretation of which is the domain of neurological diagnostics , 15. A tA typical sEMG signal is shown in Standard bipolar detection technique is assumed. Fundamental concepts pertaining to EMG signal acquisition are comprehensively summarized in . sEMG siThe diagnostic value of ECG and EEG has been established firmly in the respective medical fields of cardiology and neurology; in addition, these techniques may be conveniently used in less clinical realms such as health kinesiology (ECG) or in brain research (EEG). It is understood, of course, that these bioelectric indicators most often form part of more comprehensive measurement and evaluation schemes in addition to other clinical diagnostics data such as CT or NMR records and relevant laboratory diagnostic results. Therefore, ECG and EEG methods may certainly be considered to represent a reliable and standard component of the so-called evidence-based medicine approach.Compared with ECG and EEG, the field of clinical use of sEMG might seem less developed and standardized. sEMG was first considered and accepted as a relevant quantitative indicator in physical and rehabilitation medicine. In this field of application, sEMG forms a standard component of an assortment of biomechanical measures relevant for monitoring, evaluation, and (motor) learning in the rehabilitation context . The goaEssentially, and assuming a quantitative relationship between sEMG and muscle force [, 28–33—cMeasurement and interpretation of bioelectric signals, and of sEMG in particular, is a technical issue whereby a measurement method is applied to human subjects. Besides carrying diagnostic information, sEMG also may serve as a control variable . It can therefore be explained and understood most thoroughly—both theoretically and practically—assuming a biomedical engineering (BME) perspective. We begin this section with a short comment on BME education, both worldwide and in Croatia, focusing later on education of medical doctors, physiotherapists and kinesiologists.An inter- and multi-disciplinary approach to education is integral to the field of biomedical engineering. In Germany, between the two World Wars, and in the USA, especially after the World War II, independent university programs in the field of biomedical engineering were established. As a part of these efforts, DeClaris and Newcomb stated in the 1980s the need for biomedical engineering students to adapt traditional engineering knowledge of circuit theory toward systems theory in order to link them to physiological concepts more easily. Engineering analysis and design methods had to be adapted to the solution of problems related to biological systems . BiomediAt the beginning of the 1980s, at the Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA, for example, the undergraduate curriculum for medical doctors already featured some basic concepts in the technical (engineering) sciences such as signal and systems theory and feedback control systems theory . These cIn the current academic curricula, primarily for biomedical engineers, but also, to a lesser degree, for non-engineers , the issues of measurement and signal processing methods for all types of bioelectric signals  are successfully covered. In addition to classical mathematical signal processing methods, which are commonly taught in engineering schools, numerous advanced methods, such as data mining, neural networks, artificial intelligence, and advanced statistics, are included. Further, there is no discrimination between applicability to ECG, EEG, and sEMG in this respect. It is understood that each of these signal subtypes is treated in accordance with the specificities of its domain of application. Numerous textbooks that cover these fields are available, e.g., Sornmo and Laguna , Begg anIn Croatia, unlike in neighboring countries, no formal biomedical engineering educational programs are available to date; in contrast, in Italy and Slovenia, the BME field, including BME education, is highly developed. In Slovenia, the example of the Vodovnik group tradition from the late 1960s onward , 51 in tAt present, teaching activity in the area of biomedical engineering and physics in Croatia is accomplished mainly through a number of graduate and post-graduate mandatory and elective courses, at several university departments. In the respective courses, in addition to teachers with an engineering background , teaching staff with a biomedical background participates, including those with clinical expertise.,,,,The subject of sEMG is included in the curriculum of human locomotion study, with biomechanical approaches being pursued. At the University of Zagreb, the subject of locomotion is covered rather well, and includes courses at both the under- and the post-graduate level at several departments: the Faculty of Kinesiology, the School of Medicine, and the Faculty of Electrical Engineering and Computing, as well as at a couple of other institutions . RelevanAdequate laboratory facilities exist in Zagreb and in Pula, and, to a lesser degree, also in Split and Rijeka. Examples of undergraduate elective courses at the University of Zagreb are: Multisensor systems and locomotion,,,During undergraduate study at all medical faculties  future physicians are introduced to EMG as an electrophysiological diagnostic method and its significance in treatment and rehabilitation in the fields of taught neurology, physical medicine, and general rehabilitation, as well as pediatricsPhysiotherapists in Croatia complete the physiotherapy studies at the University of Applied Health Sciences in Zagreb  after which they receive the title “Professional Bachelor of Physiotherapy ,” abbreviated as bacc. physioth. Additional education through the Specialist Professional Study for a period of 2 years confers the title of Bachelor of Physiotherapy or Master of PhysiotherapyAt the Ph.D. level of studies, the subject of sEMG is taught primarily at the University of Zagreb , but is also included in a number of courses at other universities. Doctoral studies in kinesiology at the University of Zagreb may be taken as an example  where thEmployees of the Department for Rehabilitation and Orthopedic Devices University Clinical Hospital Zagreb participate in both undergraduate and post-graduate teaching of students of the School of Medicine, deliver classes for physiotherapists, occupational therapists, and nurses of the University of Applied Health Sciences, and collaborate in teaching delivered to students of the Faculty of Kinesiology, Faculty of Education and Rehabilitation Sciences, and Polytechnic (Tehničko veleučilište) in Zagreb. The Department further provides education for specialists in orthopedics, traumatology, and physical medicine and rehabilitation, as well as compulsory internships for B.Sc. degree students in physiotherapy, occupational therapy, and nursing. In offering education in the fields of prosthetics and orthotics, the Department collaborates with ISPO-Croatia  and Human Study e.v., and with the Polytechnic in Zagreb.A short overview of biomechanical research in Croatia, encompassing activities at the Faculty of Kinesiology and the Faculty of Electrical Engineering and Computing, University of Zagreb, and at the Peharec Polyclinic in Pula, appeared in the International Society of Biomechanics (ISB) Newsletter . The sitThere is, evidently, a striking difference between the training physiotherapists get compared to the other two categories; engineers and medical doctors. What would be needed in the first place is to have laboratory facilities better equipped . Their curriculum includes courses on anatomy, physiology and biomechanics but in general there is a lack of well-equipped laboratory facilities. So, often, practical laboratory subjects such as sEMG measurement and analysis are organized through visits to a well-equipped laboratory of a collaborating institution . So, although formally being a part of teaching program, various signal processing methods in time domain and in frequency domain can only be demonstrated and not practiced and thus acquired by students themselves. Laboratories, that are situated in other institutions  normally have a qualified professional staff including clinical engineers. Basic biomechanics and sEMG in physiotherapy schools is typically thought by a couple of teachers: a physiotherapist and an electrical or computer engineer.Our experiences, and of some of our colleagues indicate that the acceptance of physiotherapy students of modern technology is good and that with better teaching conditions they would accept these, sometimes rather requiring technical protocols, and profit from it. There is no doubt that this kind of improvement in working conditions would have positive long-term consequences on the work of future physiotherapists, and that their clinical competences would also improve. In general, at present, adequate courses and upgrades for professors of physical therapy, on the subject of sEMG and other relevant medical technology, would be useful.There is no doubt that sEMG is a valuable research vehicle. In this section we address typical applications of sEMG suitable for clinical use. Although each of these applications possesses a capacity for a clinical method, the same is being realized in various degrees, depending on methodological, financial and/or other issues. There are rather stringent requirements for a clinical method: it should possess reliability, validity, sensitivity and specificity, all features being subject to verification by appropriate clinical testing and confirmation by relevant statistics. The clinical acceptance of sEMG is a key issue addressed in this paper. We explore the contribution of sEMG data to diagnostic, evaluation, treatment, (motor) control, and/or (motor) learning procedures taking place, primarily, in hospital wards where sEMG measurement equipment is available, because these factors determine its clinical acceptance. In addition, the equipment must have a satisfactory level of user-friendliness to be accepted by the staff.In general, the neuromusculoskeletal system is amenable to a number of measurement techniques, including “muscle-centered” ones such as dynamometry, mechanomyography, ultrasound, and thermography. In the broader context, standard neurophysiological, physiological , and biomechanical methods are available. Electromyography is one of the well-established “muscle-centered” techniques. Although needle EMG (NEMG) has undisputed value in the neurological diagnostics of muscular diseases, it will not be discussed here, and we continue with the overview of sEMG applications.In 2000, The American Academy of Neurology stated that more than 2,500 original articles, reviews, and books investigating the utility, techniques used, and clinical application of sEMG had been published . As a di2, sound recordings to measure snoring, continuous video monitoring, and sEMG. One sEMG channel, usually chin or mentalis and/or submentalis, is used to record atonia during REM sleep or lack of atonia in patients with REM-related parasomnia. To assess bruxism, sEMG electrodes can be placed over the masseter. sEMG analysis of intercostal and abdominal muscles may be conducted to determine effort during respiratory events. sEMG recording for monitoring of limb muscles  is used for assessing periodic limb movements and restless legs syndrome  method involves simultaneous recording of multiple physiologic variables related to sleep and wakefulness. PSG is the most commonly used test in the diagnosis of abnormalities of sleep and/or wakefulness, and can directly monitor and quantify the number of respiratory events  and the resultant hypoxemia. In addition, it is useful in treating sleep disorders from a psychiatric or neurologic (sleep-related epilepsy) viewpoint. Assessment of sleep stages requires EEG, electrooculography (EOG), ECG, pulse oximetry, respiratory effort measurement , end tidal or transcutaneous COsyndrome , 59.In Croatia, the PSG method is routinely used in both adults and children, in neurology clinics and pediatric clinics of clinical hospital centers in Zagreb, Rijeka, Split, and Osijek. Furthermore, it is used in the clinic in which pulmonary diseases are treated of the University Hospital Center Zagreb (Jordanovac) , a pediaBiofeedback is a self-regulatory procedure through which patients are given feedback that enables them to develop control over their physiological functions through the provision of real-time data . ScientiThe first therapeutic applications of sEMG in biofeedback (sEMG-B) in psychiatry were carried out in the late 1960s. The goal of the treatment was to achieve relaxation, as a principal or adjunct mean of therapy . One stusEMG signal is the most common physiological variable monitored using biofeedback, and is used in a variety of disorders such as tension headache, chronic pain, spasmodic torticollis, and temporomandibular joint dysfunction. EEG feedback, which is also called neurofeedback, is used in attention deficit hyperactivity disorder (ADHD) and epilepsy, and is increasingly the focus of research and other applications. Other commonly monitored variables are used when the aim of biofeedback is to reduce sympathetic arousal .Efficacy ratings for biofeedback training for various medical conditions have been reported in a previous study . Based osEMG-B finds important applications in the field of rehabilitation: the signals are fed back to the patient, allowing patients to self-identify their muscle activity , 70. ThisEMG-B has demonstrated its usefulness in improving muscular torque and muscle recovery, as an addition to a conventional exercise program  such as There is indeed a large number of medical conditions for which sEMG-B can be applied. In addition to those already mentioned, spinal cord injuries and low back and neck pain can also be addressed using this tool . The essGallina et al.  introducsEMG-B is applied in clinical praxis in Croatia, in all adequately equipped physical therapy units across the country. As far as we are informed, this is maybe the most traditional use of sEMG in medicine and is successfully being applied across the country to aid in various disturbances and diseases.Although still pending clinical application, the evaluation of muscle coordination by means of sEMG signals deserves to be mentioned. This kind of application is pre-dominantly research-based in nature, and involves the measurement, processing, and correlation of myoelectric signals of several muscles that are co-active in performing a certain movement. A possible use is in sports research, where smoothed (full-wave rectified and low-pass filtered) sEMG signals that represent correlates of muscle forces may be used to quantify the degree of muscular coordination when performing a motor task, as has been performed in artistic gymnastics for example , 87. ThiAnother possible application of muscle coordination evaluation is in the control of prostheses (section Myoelectric Prostheses).Taborri et al.  performeTo the best of our knowledge, in Croatia, the above-mentioned issues are not currently being investigated, nor have they achieved clinical application at present. Our own experience includes the series of research projects investigating biomechanical and neuro-muscular aspects of complex movements, including sEMG correlates of (loco)motor skill. We have explored the issue on sportive movement patterns as a model of entrainment and skill acquisition , 87. AltA distinct area of application of sEMG signals is in evaluating local muscle fatigue. This feature is based on the inherent property of a myoelectric signal to reflect the physiological status of fatigue in a muscle during muscular work. The phenomenon was first noticed as early as 1912 , and hasFurther development of the signal processing methodology  is aimedIn Croatia, despite being rather practical for application, the sEMG technique, to our knowledge, is not yet routinely applied in clinical settings for evaluation of muscle fatigue, whether in a rehabilitation or a sports training context. We believe this is partly attributable to difficulties related to the standardization of mathematical signal processing methods to evaluate fatigue under conditions of dynamic contractions. Our own experiences include using the method in various movement patterns related to physical exercise and sports activities, as referred to before. Croatian BME group did have plans to design a practical and versatile method, supported by smart phone and suited for outdoor applications. So one may conclude that this method fails shortly to satisfy requirements of a clinical muscle fatigue evaluation method. But, we are of the opinion that perspectives are rather good.The general fields of kinesiology, biomechanics, and motor control have witnessed the widespread use of sEMG as an important indicator in the quantitative characterization of movement patterns, both healthy and pathological. In combination with kinematic and kinetic measurement data, multichannel sEMG forms a standard component of instrumental setups in motion analysis laboratories aimed at measuring human posture and movement, including gait. It can typically be found in hospitals, orthopedic wards, pediatric clinics, physical medicine and rehabilitation settings, sports medicine clinics, as well as in research institutes and university departments , 103–106In the referred literature, books, as well as in hundreds of papers, the clinical value of gait analysis is undisputedly documented, although uncertainties and limitations do exist that are well-known to the biomechanics and motor control community; however, a review of these is beyond the scope of our paper. sEMG information, which is typically obtained in the form of an 8 or 16 channel telemetrically obtained record, certainly bears its value and represents a component of a valid comprehensive gait report  Figure . In the Whether a particular gait analysis requires sEMG data is specific to the problem at hand; sometimes, only kinematic and kinetic information suffices.Critical appraisal of the situation in Croatia, however, shows that clinical gait analysis is only provided by the Peharec Polyclinic in Pula. Although the necessary equipment and expertise exist at the Faculty of Kinesiology, University of Zagreb, and despite adequate experience in research applications: kinematics and kinetics in , kinematThe modern field of design, development, and application of prostheses of extremities is highly technologically complex. Here, we do not provide a comprehensive overview, but explore some important points. In the context of myoelectric prostheses, sEMG undoubtedly plays an important role: since cybernetics was first conceptualized by Norbert Wiener, sEMG forms a natural connection between a biological and technical sub-system of a “man-prosthesis” system. Childress  illustraMotorized prostheses are typically controlled with sEMG data recorded on the residual muscles of amputated limbs. However, the residual muscles are usually limited, especially after above-elbow amputations; as a result, sufficient sEMG signals for the control of prostheses with multiple degrees of freedom cannot be obtained. Signal fusion is a possible approach that may be applied to resolve the problem of insufficient control commands, wherein some non-EMG signals are combined with sEMG signals to provide sufficient information for motion intensity decoding. One possible solution is to combine sEMG and EEG, in order to improve the control performance of the upper limb. Prosthetic hands differ in their complexity and components with some offering different grip patterns . Although myoelectric components of the prosthetic hand, wrist, and elbow are available, and various combinations of myoelectric-controlled components with body-powered components to control shoulder and/or elbow function can be created, optimal results are achieved for below-elbow amputation. For proper control of the prosthetic hand, the evaluation for possible muscle or nerve damage of the stump must be performed; then, the selection and calibration of the most effective electrode site on the clear silicone test socket fitting must be carried out using an adequate tester or sEMG analysis.With regard to the status of the field in Croatia, other parts of the former State have, in the past, made greater creative contributions to the field; these include The Belgrade Hand by Tomović  and the In Croatia, the field subsequently progressed through implementation of available technical solutions, and myoelectric prostheses have been used since the 1990s, during the rehabilitation of war amputees at the Department for Rehabilitation and Orthopedic Devices University Clinical Hospital Zagreb in Zagreb, using Otto Bock products  alone have to use these equipment.” The field of BME attains a more inter-disciplinary attitude however.Important aspects of the development of sEMG technology are multielectrode (HD-sEMG) recording techniques. Although these techniques have been available for some time already –41, theyAside from the novel electrode technology mentioned here, one has to emphasize the technological development in the field of the acquisition systems. Small, wireless amplifiers have been developed that can be integrated with the detection system and interfaced with smart devices. This, although requiring certain versatility in operation and use  enables flexible applications of measurement equipment outdoors and in different milieus.An interesting further ramification of the methods of signal detection is presented by Inzelber and Hanein  who repoAnother promising application is shown in  where HDOur concluding remark on the future of sEMG focuses on man-machine interfacing possibilities in rehabilitation technologies . There aThe two main goals of our paper were as follows: first, to evaluate the scholastic coverage and second, to explore the degree of clinical acceptance of sEMG relative to ECG and EEG. We have summarized current knowledge in the field and provided an overview of the state of the art of technology and instrumentation. Our overview has been international in its approach, as well as specifically focused on the status of the field in Croatia.The scholastic coverage, through teaching and training, of the areas discussed is only partially adequate, we believe, both worldwide as well as in Croatia. Academic training for all categories of students, i.e., physiotherapists, kinesiologists, medical doctors, engineers, consists of teaching basic knowledge on technical methods of recording, and processing the signals in question, with the goal of using these empirical data, along with other available variables and/or data, in intended research and clinical applications where it can be interpreted and used to solve a problem. Of course, when teaching engineering students, a more fundamental grasp of hardware/software knowledge is to be pursued as they as future professionals will be acting not only as users but also as designers and constructors of new equipment. The biomedical engineering approach is assumed, reflecting the inter- and multi-disciplinary nature of the field. This approach is prominent in existing curricula, as reported in section Scholastic Coverage of Bioelectric Signals, depending on the availability of appropriate laboratory facilities where signal recording equipment can be used and shown to students. The transfer of knowledge is possible, provided adequately trained staff are available. There is, of course, always room for improvement and inclusion of novel teaching tools. In addition to classical teaching methods, in a classroom and in a laboratory, online materials and courses, incorporating popular video clips for showing procedures and exercises, may be used.Clinical acceptance of both ECG and EEG methods is superior and undisputed, as briefly stated in sections On the Nature of Bioelectric Signals and Their Interpretation and Comment on Diagnostic Utility of Techniques Based on Bioelectric Signals and Their Clinical Acceptance. The degrees of clinical acceptance of the sEMG method vary according to specific applications. There are many potential applications of this technique, as has been concisely discussed in section Overview of the Clinical Acceptance of sEMG. For these methodologies to be further implemented in Croatia and other countries, certain scientific as well as economic/organizational pre-requisites must be fulfilled. The scientific pre-requisites are 2-fold, comprising a research and a teaching component. The research component comprises the potential to conduct applied, clinical studies to validate and standardize applications of sEMG in the clinical environment. To achieve this, both equipment and manpower must be available, in the form of multidisciplinary research groups capable of securing research grants. The importance of proper staffing of clinical units and biomedical institutes is to be underlined at this point. This comprises having competent leadership in these institutions which requires interdisciplinary education to pursue clinical studies with the goal of developing and introducing new clinical methods. The importance of this “human” factor cannot be overemphasized.Further, the applied component of research consists of the development of innovations (university-industry collaboration)—it is assumed that there should be a demand for such products—and presumes transfer of developed products to the market. At the University of Zagreb, Faculty of Electrical Engineering and Computing, an innovation center, namely the Inovacijski centar Nikola Tesla (ICENT)Economic and organizational issues are a matter of broader social and even political endeavors supported by the relevant State ministries of health, science and the economy.In regard to the appropriate teaching and education of primarily clinical staff, we believe that the acceptance of sEMG by the staff  is generally good. We speculate that EMG information may be more easily and intuitively interpreted by non-engineers than data obtained using technically more complex apparatus like CT or NMR. New trends in the presentation of information using intuitive and visually attractive displays will increase user-friendliness and further contribute to knowledge acquisition. However, a basic limitation is a fact that sEMG instrumentation, despite not being very expensive in comparison with many other technical methods in medicine, is still not available in all working environments where it could be useful. Through our teaching experience, we have witnessed a genuine interest in the features and potential of sEMG when introduced and explained for the first time.The limitation of our study is that the assessment of the status of the field in Croatia has not been based on objective documented data on the adoption of sEMG instrumentation across institutions. We cannot conclude whether, at the level of the State, evidence of biomedical instrumentation exists in institutions such as hospital wards, university laboratories, and research institutes. Although some initiatives have been made in this direction by the State ministries of science, education, and health, these have not been implemented, to our knowledge. The effective implementation of such endeavors would be indispensable in this context.Furthermore, we did not organize and conduct a survey among users of the equipment to assess their perceptions of the instrumentation and methodology; such data would be very informative.We have based our statements on our subjective knowledge and our academic professional contacts, and the concepts explored were defined by our fields of expertise, namely electronic and biomedical engineering and biomechanics research and teaching; research and clinical work in psychiatry; and research, teaching, and clinical work in physical medicine and rehabilitation.VM, SM, and IK conceived the manuscript. VM coordinated the activities of preparing the manuscript and mainly contributed to the biomedical engineering and kinesiology aspects. SM contributed to medical and clinical aspects, primarily in psychology, psychiatry, and neurology. IK contributed to medical and clinical aspects, primarily in physical and rehabilitation medicine, neurology, and myoelectric control. All authors contributed to the scholastic aspects, and wrote and approved the final version of 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.


We proposed a method to manipulate the surface waves with a deep subwavelength metasurface by applying resonators with interfering mode resonance. The simulation results demonstrate that a single deep subwavelength obstructed groove can effectively control the propagation of surface terahertz (THz) waves by a small step increase (< 1/20 λ) of the depth or a slight change of refractive index (Δn = 0.1). The surface waves transmitted and reflected by the single groove can be controlled periodically by increasing the groove depth or refractive index with a high efficiency owing to the mode resonance between surface spoof plasmonics modes and groove cavity modes. The generated circle resonance mode provides a new idea for the development of THz devices. Importantly, the transmitted or reflected intensity of the surface wave is also enhanced by the Mode resonance. It is a simple and effective method to operate surface THz waves and manufacture more compact integrated optical devices in deep subwavelength scale. At terahertz (THz) frequencies, various subwavelength metal structures based on spoof SPPs (SSPPs)13 have been developed to efficiently control the properties of THz fields20. Based on this, a series of new THz devices with high performance have been developed and applied in different areas, such as THz sensors23, THz spectrum and imaging detection26, and THz communication27. One of the most representative devices is the one-dimensional THz metal grating waveguide, which has been designed and fabricated into different groove shapes, including rectangular, inclined rectangular, trapezoidal, V-shaped, serrated and meniscus grooves37. If the surface grating has gradient depth42, gradient period43, and vertical or downward pyramid grooves44, it can realize the so-called "trapped rainbow" that has been proved to be a reflection rather than a real EM wave trapping46. In addition to the groove shape of the grating, the surface EM wave can also be controlled by changing the local refractive index of the dielectric layer covering the metal grating, just as a new dielectric grating is formed above the metal grating47. In fact, the change of refractive index in a groove of the metal grating itself can also significantly change the transmission characteristics of the surface wave, which will be discussed below. Furthermore, detailed understanding of the physical mechanism underlying single-groove manipulation of EM waves still needs study whether there is a covered dielectric layer or not. In this paper, it is revealed that the controlling of surface EM waves can be achieved by simply changing the depth or refractive index of a single deep subwavelength groove in the metal grating, and detailed physical explanation is given. With the increase of the groove depth or refractive index, the surface wave propagation characteristics show obvious periodic changes, which is essentially caused by the mode resonance between the surface modes and the groove cavity modes, similar to the Fano resonance50. Moreover, under the mode resonance, the grating surface will form high-intensity local standing waves, which can be applied to deep subwavelength resonators or spasers.The most important characteristic of plasmonics is to confine the electromagnetic (EM) energy and enhance the interaction between light and matter in the subwavelength ranged), width (w), and period (p). By simply adjusting the main parameters of depth (d), width (w), and period (p), it can be designed to propagate or decelerate EM waves of different frequencies, because the dispersion property of the grating is determined by theses parameters45. In addition, as previously mentioned, the refractive index (n) of the material filling in the grooves can also affect the propagation of the EM waves. To further investigate how to control the surface EM waves by the mode resonance between the grating and its single groove, a metal grating model  is marked in grey in Fig. The metal grating Fig.  has rectdel Fig.  is estab45. Hence, a source of 3.75 THz is used to excite the propagating surface wave, which propagates smoothly along the grating until it encounters the special grey groove  or refractive index (Δn). The intensity of electric field at localized points could be larger than “1” because of the joint contribution of mode resonance between plasmonic surface mode and groove cavity mode, and the interference between reflection surface wave and incident wave. For a grating working as a normal waveguide, the monitored intensity values at the reflection and transmission points are almost same and far below “1” as shown in Fig. n = 0, Δd = 0 μm, the grey groove is exactly same with other grooves, and the grating becomes a normal grating. Therefore, the transmission and reflection intensity are all approximately equal to “0.4”, which is a base intensity of the surface wave. When the reflection gradually increases and the transmission gradually weakens, the measured reflection intensity starts to be greater than “0.4”, while the transmission intensity is gradually less than “0.4”. Hence, the reflection and transmission intensity value are very different. It is also the reason why the scale bar value in Fig. The cutoff frequency of a metal grating with period of 20 μm, width of 10 μm and depth of 10 μm is lower than 4.6 THzn = 0 (n = 1)     Fig. a, the ch.5) Fig. c, the ch μm Fig. b, the reμm) Fig. d, a serid = 32 μm and transmission valley at Δd = 36 μm, respectively for the surface wave (Δn = 0). It means that the excited surface wave with frequency of 3.75 THz can be propagated along the surface grating and transmitted over the single groove . However, when Δd = 36 μm, the propagated surface wave is almost totally blocked , and Fig. d = 36 μm), respectively. For Δd = 32 μm, there are no resonance between the surface spoof plasmonics mode and groove cavity mode, and the metasurface works as a high efficient waveguide. The plasmonics mode is propagated along the surface and coupled from groove to groove . Therefore, the propagated surface wave with frequency of 3.75 THz can be transmitted when Δn = 0.5, but blocked when Δn = 0.6 . Importantly, the transmitted or reflected waves are all enhanced to higher intensity than the normal surface waves by the mode resonance, which provide a new and easy way to obtain the controllable surface waves with enhanced intensity. If we want to use it for the refractive index sensing, the sensing sensitivity (Δλ/Δn) can be estimated by the transmission curves at different refractive index as shown in Fig. n = 1.5) and circle dotted red line (n = 1.6) is the existence of the transmission peak from wavelength of 78 μm to 82 μm. Hence, Δλ here could be the peak width (~ 4 μm). The refractive index sensing sensitivity (Δλ/Δn) is approximately 40 μm/RIU, which is already a competitive value comparing to typical sensors53.Similarly, the two points marked in Fig. n = 0, Δd = 36 μm) at around 3.75 THz (corresponding to wavelength of 80 μm) is presented in Fig. At last, the transmission spectrum of the single obstructed groove (ΔIt has been demonstrated that the propagation characteristics of surface waves can be efficiently controlled by a single deep subwavelength groove with only 4 μm depth changing (< 1/20 λ) or refractive index changing of 0.1. We can use a few or more special grooves of different depth or refractive index located at different positions to manipulate the EM waves of different frequencies by the mode resonance between the plasmonic mode and groove cavity mode. The physical reason is the new circle resonance mode generation. It provides a new idea for the development of new THz devices. In addition, the intensity of the transmitted or reflected waves are all enhanced by the mode resonance, which is important for high sensitivity detection. From the application point of view, the depth or refractive index of the single groove is easy to change, which can provide a simple scheme to efficiently control the transmission characteristics of the EM wave at the deep subwavelength scale. Hence, it can further promote the development of THz compact devices and integrated technology.


To evaluate implantation potential of cleavage-stage embryos cultured in medium containing 2 ng/ml granulocyte–macrophage colony-stimulating factor (GM-CSF) versus control medium, according to embryo morphological quality and then transferred on day 3.N = 286) or double-embryo transfer of two embryos with equivalent morphological quality . Implantation rate and live birth rate were assessed according to category of morphological embryo quality on day 3.Explorative secondary data analysis of a multicenter, randomized, placebo-controlled, double-blinded prospective study of 1149 couples with embryo transfer after IVF/ICSI. This analysis includes a subgroup of 422 subjects with either single-embryo transfer , and for embryos with less than 6 cells at day 3 this same rate was: 7.4 ± 3.3% (GM-CSF) versus 12.0 ± 4.0% (control) .Culture with GM-CSF did not increase the implantation rate for embryos classified as poor quality. A trend towards greater benefit of GM-CSF on implantation and survival until live birth for top-quality embryos (TQEs) compared with poor-quality embryos was observed, although not statistically significant. For TQEs, the percentage of transferred embryos resulting in a live born baby was: 40.9 ± 5.3% (GM-CSF) versus 30.5 ± 4.6% (control) (This exploratory analysis is consistent with GM-CSF protecting morphologically normal embryos from culture-induced stress and does not support an effect of GM-CSF in rescuing poor-quality embryos.ClinicalTrials.gov identifier: NCT00565747. Although current in vitro conditions for culture of human embryos attempt to mimic the in vivo environment, it is generally recognized that the achieved conditions are suboptimal, and ideal culture media for in vitro development of human embryos are yet to be developed , 2.In the female reproductive tract, several of the cytokines and growth factors secreted by the lining epithelium of the human fallopian tube and uterus have demonstrated effects in promoting normal embryo development and supporting embryo implantation . GranuloThe protective effects of GM-CSF identified for murine embryo culture include enhanced embryo viability by reduced cellular stress response and apoptosis , 9, and A prospective randomized, placebo-controlled, double-blinded multicenter trial reported by Ziebe et al. , for whiRegarding the possible mechanisms involved, a report by Karagenc et al. in mouse embryos  suggesteIn this explorative secondary analysis of data generated during the multicenter prospective randomized trial reported by Ziebe et al. , we aimeN = 286) or a double-embryo transfer (DET) (N = 136) of two embryos of comparable morphological quality. Women transferred with two embryos of different morphological quality (N = 106) were excluded from this investigation.This is an explorative secondary analysis of a subset of data from a previously reported prospective randomized, placebo-controlled, double-blinded, multicenter trial  . BrieflyDetails on study design and randomization procedure have been described previously . BrieflyEmbryos were categorized by morphological score, with ‘Abnormally Developed’ embryos (ADE) having < 6 cells at 68 h post-insemination, ‘Normally Developed’ embryos (NDE) having ≥ 6 cells and ≤ 20% fragmentation at 68 h post-insemination, without fulfilling the criteria for Top Quality Embryo (TQE); and TQE having 4–5 cells at 44 h post-insemination, ≥ 7 cells at 68 h post-insemination, ≤ 20% fragmentation, equally sized blastomeres with < 25% difference, and no signs of multinucleation.N = 422 subjects). Odds ratios (ORs) for the effect parameters  were analyzed with the use of a logistic regression model (SAS software version 9.2) with treatment group (GM-CSF/control) as main factor, and covariates including age, smoking status and number of oocytes collected. Age, BMI and number of oocytes collected were compared between GM-CSF and control group using the t test. All categorical comparisons were performed using Fisher’s exact test.The effect of GM-CSF on embryo implantation was investigated by exploratory statistical analysis to evaluate trends with regard to embryo implantation rate and survival until live birth when comparing embryos of comparable morphology. Data analysis were performed for a combined group of women with single-embryo transfer or double-embryo transfer with embryos of equivalent morphological quality  cultured in GM-CSF or control medium are presented in Tables P = 0.24; odds ratio [OR] 1.43, 95% confidence interval [CI] 0.79–2.59).The clinical success rates after IVF/ICSI and embryo culture and transfer in GM-CSF and control medium, respectively, according to the morphological category of the embryo(s) transferred, are shown in Table P = 0.26; OR 0.53, 95% CI 0.17–1.61). Moreover, there was no effect of GM-CSF on the group of NDE’s : with the percentage of transferred embryos resulting in a live born baby being 22.9 ± 4.8% (GM-CSF) versus 22.4 ± 4.9% (control) .However, no beneficial effect of culture with GM-CSF was observed in the remaining subgroups of abnormally (ADE) and normally developed embryos (NDE). In contrast, a tendency to decreased implantation potential was observed for the group of embryos with poor morphological quality (ADE) when cultured in GM-CSF medium, with the percentage of transferred embryos resulting in a live born baby being 7.4 ± 3.3% (GM-CSF) versus 12.0 ± 4.0% (control) .Here, we report an exploratory secondary analysis investigating a subgroup of patients participating in a multicenter prospective randomized trial reported by Ziebe et al. . The subAlthough statistical significance was not reached due to insufficient power for this exploratory subgroup analysis, it was a consistent observation for all success parameters both on the embryo level and patient level that the presence of GM-CSF reduced favorable outcomes in the poor embryo quality group; while in the highest embryo quality group, GM-CSF exhibited favorable effects. In the intermediate group, no effect of GM-CSF was observed on the success parameters. These data suggest that the improved implantation rates and progression to live birth found by Ziebe et al.  is princImportantly, this analysis alleviates any concerns that pro-survival cytokines may cause embryos otherwise destined for demise, to survive. It has been suggested that GM-CSF might rescue developmentally compromised embryos, potentially resulting in implantation and ongoing development of embryos that would not have survived in the absence of cytokine . Our expThe current data also imply that beneficial effects of GM-CSF cannot be attributed to rescuing human embryos from poor or highly stressful culture conditions , as all Our rationale for this analysis was to search for evidence of GM-CSF effects acquired by the cleavage-stage embryo during culture. Effects of GM-CSF that are evident at blastocyst stage, particularly reduced incidence of apoptosis and elevated cell number , might bThis result adds to previous evidence that exposure of human embryos to GM-CSF in culture does not increase the likelihood of stillbirth, perinatal death, congenital abnormalities or abnormal birth weight of singletons and twins when compared with control medium without GM-CSF . The res


Hand hygiene in refugee camp settlements remains an important measure against diarrhoeal infections. Refugee settings are characterised by overcrowding and inadequate access to water and hygiene facilities which favour proliferation of faecal-oral diseases. Handwashing with soap and water is therefore an effective way of preventing such diseases. Despite this knowledge, there is limited information about access to functional handwashing facilities in these settings and associated factors in Uganda.  Quantitative data were collected from 312 refugee households in Rhino Camp Settlement, Northwestern Uganda, using a semistructured interviewer-administered questionnaire. A modified Poisson regression was used to obtain prevalence ratios (PRs) and 95% confidence intervals (CIs) for the determinants of access to a functional handwashing facility among refugee households. All analyses were performed using STATA 14.0 statistical software.  Of the 312 households, 123 (39.4%) had access to a handwashing facility, but only 72 (23.1%) of households had handwashing facilities that were functional. Duration of stay in the camp exceeding 3 years ) and history of receiving home-based education on hand hygiene ) were independent predictors of access to a functional handwashing facility.  Access to functional handwashing facilities among the refugee households was low. Our findings highlight the need for more and continued handwashing promotional programs, most especially among newly arrived refugees in the camp. Hand hygiene in emergency settings remains an important measure against the risk of spread of communicable diseases. Evidence from systematic reviews and clinical trials indicate that handwashing with soap can reduce risk of diarrhoeal infections by 30% to 50% –3 and riIn refugee settings, the situation is likely to be worse since there is substantial disruption of habitual and cultural norms thereby potentially altering refugees' practices such as handwashing . In suchIn Rhino camp settlement, Uganda, aid agencies such as the International Federation of Red Cross, Uganda Red Cross Society, and World Vision have promoted water, sanitation, and hygiene by ensuring universal access to improved water sources. However, this water supply is often insufficient. Consequently, the limited water tends to be prioritized for drinking and cooking, while hygiene gets short shrift, especially among people who still struggle to understand the connection between hygiene, sanitation, and health . MoreoveCurrently, it is not known what proportion of households actually have access to these handwashing facilities at their premises. The functionality of these facilities in terms of availability of sufficient water and soap is not known yet. This information could act as a proxy for handwashing behaviour. In our study, we assessed access to functional handwashing facilities and associated factors among refugee households in Rhino settlement camp in Northwestern Uganda. The identification of the determinants of access to handwashing facilities can guide the design of handwashing promotional programs. This is expected to reduce disease burden, health care expenditure at individual, organisational and societal level and consequently improve health and increase productivity among refugees.2 [https://data2.unhcr.org/en/documents/download/64539. These refugees are mainly from South Sudan and are of diverse ethnic backgrounds, namely, Dinkas, Kuku, Nuer, Kakwa, Madi, and Siluk. The study units for this study were households within the refugee settlement, while adults aged 18 years and above residing in the households within the camp formed the study population.This was a cross-sectional, descriptive study among refugee households in Rhino camp settlement. Rhino camp settlement lies in the Northwestern corner of Uganda, 520 kilometres (km) from Kampala city and only 80 km from the South Sudan border. The camp has existed since 1980s, but in August 2017, the settlement was expanded due to influx of refugees following the South Sudan Civil War and currently hosts over 116,000 refugees. The expansion resulted in establishment of Omugo zone extension area [2 . The mapA total of 14 clusters from the 7 administrative zones (2 clusters per zone) were selected by simple random sampling. For this study, a cluster was taken to be a village which is the lowest local administrative unit in Uganda. Selection of clusters was done by entering all the names of the villages for each zone into a computer and with the help of the random function in Microsoft Excel.K for each village was calculated by dividing the total number of households in the village by the number of households targeted for inclusion in the study (23). The local leader provided the information on household population of his/her village.From each selected cluster, a total of 23 households were selected using a systematic random sampling technique leading to an estimation of 322 households. The sampling interval Kth household from the centre of the village in the Northern direction was considered until the village boundary was reached and the same pattern was repeated in different directions clockwise.A compass was used to identify the starting direction. The research team, guided by the village leader, located the centre of the village and then used a compass to identify the northern direction as the starting direction for household interviews. Every The research assistants then interviewed only one participant per sampled household. In cases where there was more than one eligible participant, simple random sampling was used to select a respondent to answer the questionnaire. This was done by randomly picking from a bag a folded ballot paper with a number corresponding to the participant's name who was then interviewed.Data were collected in August 2018 using an interviewer-administered semistructured questionnaire. The original English questionnaire was translated to Arabic, the local language spoken by the refugees. Responses were recorded on the English questionnaire. Before using the tool, the Arabic tool was back translated to English to check for consistency. Data were collected on sociodemographic characteristics, water sources, hand hygiene facilities, and knowledge related to hand hygiene and health. The questionnaire was developed based on reviewed literature on handwashing in refugee settings , 13, 14.where appropriate) were performed for respondent characteristics , accessibility to improved water sources, health education sessions on hand hygiene, and access to handwashing facilities. The outcome variable  was dichotomized and was derived as follows. A household was considered to have a functional handwashing facility if (1) there was a handwashing facility or specific place set for handwashing and (2) cleansing materials  and water were available at that facility/specific place. To assess the association between access to a functional handwashing facility and each explanatory variable, we applied a generalized linear model of the Poisson family, with logarithm as the canonical link function and applying robust error variance to obtain the crude prevalence ratios (PRs) and their corresponding 95% confidence intervals (CIs). Variables that had threshold probability p value ≤0.2 in bivariable models were all added into the multivariable regression model and a stepwise backward elimination method was used until only significant predictors were retained in the model. Crude PRs and adjusted PRs were reported. All p values were two-sided and considered significant if less than 0.05.Data were entered in Epidata 3.02  and analysed using STATA 14.0 statistical software . Descriptive analyses such as frequencies, proportions, and means . Majority of the respondents were females 70.8% (221/312) and had lived in the camp for less than 3 years 74.0% (231/312). Three quarters 67.9% (212/312) were married and nearly half 47.1% (147/312) had no formal education .In Rhino camp settlement, all households had access to improved water sources. The most popular of these water sources were public tap stands 205 (65.7%) followed by boreholes 104 (33.3%). Overall, a total of 123 households (39.4%) had access to a handwashing facility at theirpremises. Only 72 households (23.1%) had handwashing facility with both soap/ash and water. These households, therefore, were considered as having access to a functional handwashing facility .p value <0.001). Households that had received home-based education sessions in the last six months had higher likelihood of having a functional handwashing facility compared to those who had not received home-based education session in the same time period , and p value = 0.021) , and = 0.021) .This study sought to understand household access to functional handwashing facilities and associated factors in Rhino camp settlement in Arua district, Northwestern Uganda. Our findings revealed that household access to functional handwashing facilities was low. Furthermore, household access to functional handwashing facilities was associated with the duration of stay in the camp and history of receiving home-based education on hand hygiene.Our study findings showed that approximately 3 in 10 households had access to a handwashing facility supplied with soap and water. In contrast, a study in refugee camps in South Sudan found that handwashing facilities existed in more households (64.3%) and (34.9%) of the handwashing facilities had water and soap . In our Insufficient soap can be a major barrier to handwashing, often resulting into using the same piece of soap for multiple purposes which may include laundry and bathing . In our In multivariable analysis, length of stay in the camp showed a significant association with household access to a functional handwashing facility. Households that had stayed for at least 3 years in the settlement camp were more likely to have access to a functional handwashing facility than those that stayed for less than 3 years. This is in line with evidence that suggests that people who have lived longer in refugee camps are expected to have more frequent exposure to hygiene promotion activities and can adopt good hygiene practices such as possession of functional handwashing facilities . In contOur study also indicated that households that received basic home-based education sessions on handwashing within 6 months preceding the survey were more likely to have access to functional handwashing facilities. Health education programs on hygiene have shown preeminence over only investing in handwashing facilities in improving handwashing behaviour, and this is due to the belief that behaviour is learned and consolidated . A studyA limitation of our study is that we assessed access to functional handwashing facility, which though reliable, is only a proxy indicator and does not give a more accurate estimate for handwashing practice. Better estimates can be obtained from long-term direct observation methods on handwashing. However, this study provides useful insights into access to functional handwashing facilities and its associated factors among refugee households in Northwestern Uganda. Areas of further research could include conducting studies on handwashing practices using direct observation methods and for an extended period.Access to a functional handwashing facility among refugee households was low. In order to improve access to a functional handwashing facility, there is need to ensure availability of soap at the sites for handwashing and, more critically, home-based health education programs on hand hygiene should be intensified. Longer duration of stay in the camp was also associated with access to a functional handwashing facility. We suggest continuous home-based education programs on hand hygiene, particularly focussing on newly arrived refugees in the camps.


Handwashing is important in preventing infectious diseases like COVID-19. The current public health emergency has required rapid implementation of increased handwashing in the general public; however, rapidly changing health behavior, especially on this scale, is difficult. This study considers attitudes and affective responses to handwashing as possible factors predicting COVID-19 related changes to handwashing behavior, future intentions, and readiness to change during the early stages of the pandemic in the United States. Income was explored as a potential moderator to these relationships. To explore these issues, data from 344 community participants were analyzed. Results indicate that stronger affective responses toward handwashing relate to increases in handwashing since the outbreak of COVID-19, and both attitudes and affect uniquely predict handwashing intentions. Income significantly moderated the relationship between affect and readiness to change. Those with low income were more influenced by both affective responses and attitudes. These results suggest messages targeting both cognitions and affective responses are needed to increase the handwashing behavior during a global pandemic and these variables are critical in increasing readiness to change in low-income individuals. Due to the rapid spread of the SARS-CoV-2 virus, it was critical that Americans quickly implemented health behaviors such as social distancing and frequent handwashing. This implementation is, perhaps, easier said than done, as rapid implementation of behavior change is notoriously difficult to achieve in many health domains. With the backdrop of a global pandemic, it was unclear how Americans would response to this need for behavior change, or what factors may be influential. Handwashing was not only among the first behaviors recommended to the public, remaining visible to the public throughout the course of the pandemic, but it is also central to the prevention of the spread of SARS-CoV-2 . As suchFactors related to behavioral intentions and change have been widely explored, however the rapidity of the change due to the pandemic in the United States is unprecedented, and it is unclear what factors will be most influential on behavior and intentions. Cognitive variables, such as attitudes, have long been established as consistent predictors of behavioral intentions in models such as the Theory of Planned Behavior , and attAlongside attitudes, affective responses to health behaviors have recently been acknowledged as important predictors of health behaviors . For exaa priori hypotheses were made and separate analyses were run for each study.While attitudes and affect are expected to drive handwashing behavior, intentions, and readiness to change, it is possible that the influence of these predictors varies with important social factors. One possibility, explored here, is that income moderates these effects. It is well documented that income and factors related to income are determinants of health . EmerginN = 344) were recruited from Prolific, an online participant recruitment system at the end of March 2020, just as stay home and shelter in place orders began to be issued in many states. Participants ranged in age from 18 to 74 , and were fairly evenly distributed by gender, with 54.1% identifying as women, 43.9% identifying as men, and 1.5% identifying as another gender or preferring not to disclose their gender. Participants lived in the United States at the time of survey completion and represented 44 of the states. Participants were 68% White, 16% Asian/Asian-American, 5% Black, 5% Latinx, and 6% two or more races. Participants were compensated for participating in the study.Community participants . As such, the five items were averaged together to create a single bipolar affect toward handwashing measure.Five items that assessed affective responses related to handwashing were measured in order to capture the strength of these affective responses. Items included both positively and negatively valanced affective states, including anger, pride, guilt, annoyance, and feeling in control. Example items include “I am angry when others do not wash their hands” and “I am proud of washing my hands.” Items were scored on a 7-point Likert scale ranging from COVID-19 related handwashing behavior change was calculated by asking participants to self-report the number to times they washed their hands daily before the outbreak of COVID-19 and then after the outbreak of COVID-19. Both items allowed participants to enter a numeric response into an open response item. Analyses were conducted on the post-COVID-19 handwashing behavior while controlling for self-reported pre-COVID-19 handwashing behavior.α = 0.80).Handwashing intentions were assessed using six questions that targeted the intention to wash one’s hands in scenarios recommended by health organizations, such as “after blowing your nose, coughing or sneezing” and “after touching surfaces outside of the home, including money.” The final item asked about intention to wash one’s hands for at least 20 s each time. The scale demonstrated a high level of internal consistency  of the relationship between affect and readiness to change, F = 7.71, p < 0.001, R2change = 0.02. Specifically, stronger affective responses resulted in greater readiness to change for low-income Americans, but not high-income Americans  can be found at: The studies involving human participants were reviewed and approved by University of Toledo Institutional Review Board. The patients/participants provided their written informed consent to participate in this study.KC was the lead writer of the original draft, involved in the conceptualization and developing the methodology of the project, and responsible for the collection and formal analysis of the data and visualizing data. JM was involved in the conceptualization of the project and the interpretation of results, and contributed to the original draft and review and editing. KF was involved in the conceptualization and methodology of the project and editing and revision process, aided in the development of project materials, and provided supervision. AG was involved in the supervision, conceptualization, and methodology of the project, aided in the development of project materials and formal data analysis, contributed to the original draft, and provided editing and revisions. All authors contributed to the article and approved the submitted version.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.


Higher hemopexin was associated with higher CSF amyloid (implying decreased brain amyloid deposition), improved hippocampal metabolism and cognitive performance. Meanwhile, hemoglobin subunits were associated with increased CSF tau (implying increased brain tau deposition). When dichotomizing individuals with mild-cognitive impairment into stable and converters to Alzheimer's disease, significantly higher baseline hemoglobin subunits were observed in the converters compared to non-converters. Heme/iron dyshomeostasis is an early and crucial event in AD pathophysiology, which warrants further investigation as a potential therapeutic target.Brain iron dyshomeostasis is a feature of Alzheimer's disease. Conventionally, research has focused on non-heme iron although degradation of heme from hemoglobin subunits can generate iron to augment the redox-active iron pool. Hemopexin both detoxifies heme to maintain iron homeostasis and bolsters antioxidant capacity via catabolic products, biliverdin and carbon monoxide to combat iron-mediated lipid peroxidation. The aim of the present study was to examine the association of cerebrospinal fluid levels (CSF) hemopexin and hemoglobin subunits (α and β) to Alzheimer's pathological proteins (amyloid and tau), hippocampal volume and metabolism, and cognitive performance. We analyzed baseline CSF heme/iron proteins (multiplexed mass spectrometry-based assay), amyloid and tau (Luminex platform), baseline/longitudinal neuroimaging  and cognitive outcomes in 86 cognitively normal, 135 mild-cognitive impairment and 66 Alzheimer's participants from the Alzheimer's Disease Neuroimaging Initiative-1 (ADNI-1) cohort. Multivariate regression analysis was performed to delineate differences in CSF proteins between diagnosis groups and evaluated their association to amyloid and tau, neuroimaging and cognition. A  The amyloid cascade hypothesis remains the major framework for explaining the pathophysiology of Alzheimer's disease (AD) , in a cohort comprising cognitively normal (CN), MCI and AD  from the Alzheimer's Disease Neuroimaging Initiative-1 (ADNI-1) study were included (http://adni.loni.usc.edu/). This group comprised 86 CN participants, 135 participants with MCI and 66 participants with AD. All the participants underwent apolipoprotein E (APOEε4) genotyping. ADNI uses serial clinical and neuropsychological assessments, imaging and CSF biomarkers to monitor progression of MCI subjects to AD. Written informed consent was obtained and approved by the institutional review board at participating centers for the use of human data in ADNI database. Data used in the preparation of this article is available on the ADNI database (adni.loni.usc.edu).Participants , the National Institute of Biomedical Imaging and Bioengineering (NIBIB), the Food and Drug Administration (FDA), private pharmaceutical companies and non-profit organizations, as a $60 million, 5-year public-private partnership. The primary goal of ADNI has been to test whether serial magnetic resonance imaging (MRI), PET, other biological markers, and clinical and neuropsychological assessment can be combined to measure the progression of MCI and early AD. Determination of sensitive and specific markers of very early AD progression aids researchers/clinicians to develop new treatments and monitor their effectiveness, and identifies suitable participants for enrolment clinical trials. The Principal Investigator of this initiative is Michael W. Weiner, MD, VA Medical Center and University of California San Francisco, with many co-investigators from a broad range of academic institutions and private corporations. Subjects have been recruited from over 50 sites across the U.S. and Canada, for up-to-date information, please see Enrolled subjects in the ADNI-1 cohort were 55–90 years of age, accompanied by a study partner able to provide independent evaluation of recruited participant's functioning, and speak either English or Spanish fluently. Participants must have a Hachinski Ischaemic score ≤4, geriatric depression scale <6, visual and auditory acuity adequate for neuropsychological testing, six grades education or work history and not enrolled in other trials or studies. Individuals on specific psychoactive medications e.g., narcotic analgesics, neuroleptics, anticholinergic agents, antiparkinsonian medications, investigational drugs, benzodiazepines, antihypertensive agents with frequent central nervous system side-effects, antidepressants, within the 4 weeks prior to screening were excluded. Individuals with any serious neurological disease other than AD, any history of brain lesions or brain trauma, were also excluded.CN subjects must have no significant impairment in their cognitive domains or impaired activities of daily living, with a mini-mental state examination (MMSE) score between 24 and 30, a CDR of 0, non-depressed, no MCI and non-demented. The age range of CN individuals was matched to that of MCI and AD subjects. MCI subjects must also have an MMSE score between 24 and 30 but have a memory complaint, experience objective memory loss measured by education adjusted scores on Wechsler Memory Scale Logical Memory II, a CDR of 0.5, although no significant impairment in other cognitive domains and preserved activities of daily living, and free of dementia. The AD cases included in the study had MMSE scores between 20 and 26, CDR of 0.5 or 1.0 and met National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer's Disease and Related Disorders Association criteria for probable AD (NINDS–ADRDA).http://www.adni-info.org). ADAS-Cog13 is a 13-item scale used for assessing learning, memory, language production and comprehension, constructional and ideational praxis, orientation, has number cancellation and delayed free recall tasks. The word recall test was administered first, and the word recognition task given at the end with other cognitive tasks given in between. The two-word memory tasks were separated so that the risk of individuals confusing words from the two tasks was minimized. Objective testing was followed by subjective clinical ratings of language ability and aptitude of the participant to remember test instructions .All subjects underwent MMSE, Rey Auditory Verbal Learning Test (RAVLT) and Alzheimer's disease assessment scale 13 (ADAS-Cog13). MMSE measures orientation, attention, memory  and language. RAVLT tests episodic verbal memory by assessing an individual's ability to acquire a list of 15 words over five trials. The test comprised a short-delay recall trial presented after a distracter list, and a 30 min long delay recall trial, and finally by a yes/no recognition trial (http://adni.loni.usc.edu/). Although a neuropathological diagnosis is required to confirm diagnosis of AD, studies have shown high sensitivity and specificity using neuroimaging and neuropsychological assessments for determining MCI conversion to AD  and total tau (ttau) were measured using the Luminex platform as described previously  was used to calculate hippocampal volumes and described previously  sequence . The FDG-PET images were pre-processed according to standard ADNI procedures with frames co-registered, averaged and reoriented along the anterior-posterior commissure line and resliced to a 1.5 mm isotropic voxel space. Each PET image was spatially normalized to Montreal Neurological Institute (MNI) space and the mean hippocampal FDG uptake  measured .[p-value correction as in the case of univariate analysis, hence the error rate equals the significance level. Moreover, MANCOVA assesses the pattern between multiple dependent variables which is not detected in ANCOVA analysis, leading to greater statistical power to locate differences between groups.Statistical analysis was performed using SPSS IBM version 24.0. GraphPad Prism 8.4.2  was used to compute heatmaps. We used MANCOVA modeling to assess the differences in CSF proteins  across diagnostic groups, with age, sex and APOEε4 genotype included as covariates. The advantages of using MANCOVA extends the capabilities of ANCOVA by enabling assessment of multiple dependent variables simultaneously. MANCOVA require additional We also repeated the MANCOVA analysis (as above), but this time to assess differences across CSF biomarker negative CN, and biomarker positive MCI and AD subjects, rather than solely across diagnostic groups. Subjects were stratified according to the previously published CSF total tau/Aβ ratio threshold ratio  and cognitive  measures. We also examined the association of CSF proteins with longitudinal changes in neuroimaging and cognitive measures, which were computed using the following equation:We extended the findings from the partial correlation analysis by conducting multivariate regression modeling to confirm the associations. Since follow-up times were different between subjects, follow-up time was included as a covariate. A minimal model-based approach was utilized using the Akaike information criterion (AIC) and Bayesian information criterion (BIC) to yield the most appropriate fitted model.MCI is known to be a heterogeneous group, with differing rates of progression to AD, these individuals were a focus of this study. We grouped MCI subjects according to their CSF biomarker status, to examine relationships between biomarker status and levels of HPX, Hbα, and Hbβ. We also compared baseline HPX, Hbα, and Hbβ levels between MCI-c and MCI-nc, but only in those MCI individuals who were CSF biomarker positive, to determine if they are altered prior to conversion from MCI to AD. Two-tailed t-tests were computed to assess the differences.p-value ≤ 0.05 was considered significant for MANCOVA and multiple regression analysis. A false discovery rate (FDR) correction set at Q of 5% was then applied to correct for multiple comparisons for t-tests (corrected p ≤ 0.0397) and partial correlation analysis (corrected p ≤ 0.036).For statistical analysis, we tested data satisfied assumptions by checking for collinearity, normal distribution of residuals, maintenance of homoscedasticity and normality of error terms, using Levene's test, Box's test of equality of covariance matrices, histogram, P-P and Q-Q plots. The regression standardized predicted values were plotted against standardized regression residuals and if points were found to be equally distributed with no obvious pattern, homoscedasticity was confirmed. Aβ, ptau and ttau were log-transformed to ensure normality. Values were quoted as mean ± standard deviation (S.D.). A n = 273) while the remaining were white Hispanic (n = 4), black non-Hispanic (n = 10) and asian non-Hispanic (n = 3). The age ranges for CN (75.70 ± 5.54 years), MCI (74.69 ± 7.35 years) and AD (74.98 ± 7.57 years) were similar. The CN group had 49% females, MCI had 33% while AD, 29%. The years of education was similar in CN (15.64 ± 2.97), MCI (16 ± 2.97) and AD (15.11 ± 2.96). APOEε4-positivity was observed in 24% of CN, 53% of MCI and 71% of AD.Of our ADNI cohort, most participants were white non-Hispanic (p = 0.836), Hbα (p = 0.814), and Hbβ (p = 0.997) were similar in CN, MCI and AD , Hbα  or Hbβ  between diagnostic groups. Expectedly, CSF Aβ levels (p = 2.369 × 10−7) were significantly decreased with advancing disease (CN > MCI > AD). CSF ptau (p = 6.051 × 10−8) and ttau (p = 1.492 × 10−7) were significantly increased in MCI and AD. Meanwhile, hippocampal atrophy (p = 6.587 × 10−16) and glucose hypometabolism (p = 1.438 × 10−17) were pronounced in MCI, with further worsening observed in AD. The cognitive performance measured by MMSE (p = 3.255 × 10−48), RAVLT (p = 7.620 × 10−34) and ADAS-Cog13 (p = 3.231 × 10−43) was significantly impaired with advancement in disease.The baseline levels of CSF HPX  and CSF Aβ  levels; and improved longitudinal changes in glucose metabolism , MMSE  and RAVLT  scores. A positive correlation was observed between CSF Hbα and Hbβ levels . High levels of CSF Hbβ were related to lower levels of CSF Aβ but at a non-significant level . Higher levels of CSF Hbα  and Hbβ  were significantly associated with increased levels of CSF ptau. CSF HPX , Hbα  and Hbβ  levels were not significantly associated with CSF ttau.Using partial correlation analysis adjusted for diagnosis and APOEε4 genotype , 2, highInitially, we devised our model using CSF HPX, Hbα, and Hbβ as independent variables, with baseline CSF APOE, Aβ, ptau and longitudinal changes in hippocampal metabolism, MMSE and RAVLT scores as dependent variables. The ttau levels, baseline scores of neuroimaging and cognitive performance were not included as they showed no association with either CSF HPX, Hbα or Hbβ based on partial correlation analysis. Further, as CSF Hbα and Hbβ showed significant positive correlation, multicollinearity was detected, which renders the relative strength of predictor variables and their interaction effects unreliable. To ensure absence of collinearity, we selected CSF Hbβ and excluded CSF Hbα as the former was a better fit for the model. In the process, the model fit was improved from an AIC of 492.719 and BIC of 602.504 to 471.186 and 548.035, respectively. To ensure the central theorem hypothesis  was maintained, we had to remove longitudinal change in the MMSE variable. The finalized model included CSF HPX and Hbβ as predictors to the model which included CSF APOE, Aβ, ptau, longitudinal changes in hippocampal metabolism and RAVLT as dependent variables. The finalized model had an improved AIC of 285.768 and BIC of 351.638, confirming the achievement of a best-fitted model.p = 0.002) and CSF Aβ  levels, improved longitudinal hippocampal glucose metabolism  and RAVLT performance . Meanwhile, higher levels of CSF Hbβ were associated with deteriorated longitudinal hippocampal glucose metabolism  and non-significantly decreased CSF Aβ , and significantly increased ptau  levels.Higher levels of CSF HPX were associated with increased CSF APOE . However, CSF Hbα and Hbβ were higher in biomarker-positive MCI individuals compared to those that were MCI that was biomarker-negative .Since MCI represents a heterogenous group at heightened risk of converting to AD, we assessed baseline levels of CSF HPX, Hbα and Hbβ and determined their relationship with disease progression. Baseline CSF HPX levels were significantly lower in MCI biomarker-positive individuals compared to MCI biomarker-negative individuals  CSF APOE, (2) CSF Aβ levels, (3) hippocampal glucose metabolism and (4) cognitive performance. Moreover, CSF Hb subunits were significantly higher in MCI-c compared to MCI-nc and were associated with decreased CSF Aβ and increased CSF ptau. The present CSF study extends our recent plasma study  aggregation, prolonging peroxidation and perpetuating neuronal oxidative stress . In our previous study (Ashraf et al., It is worth noting that the correlations in this study are weak but significant. Since correlational analysis does not equate to causation, further studies need to be undertaken, including measurement of HPX and Hb subunits in a separate set of samples, preferably with matched CSF and plasma samples, to assess the reproducibility of the associations observed in the ADNI dataset. Further investigations are required to determine a possible causal relationship between HPX and AD pathogenesis. Importantly, studies should temporally evaluate CSF HPX and Hb subunits in prodromal AD subjects to understand how the levels of proteins fluctuate with disease progression. Since we did not assess the functional status of HPX, it is possible that there is loss of the scavenging capabilities of HPX due to oxidative modification (Hahl et al., We did not find significant alterations in CSF protein levels between CN, MCI and AD. This could be attributed to local regional variability in their levels, particularly in disease-affected brain areas that may not lead to changes in CSF proteins levels, since CSF levels results from the summed metabolism of the whole brain.Most importantly, iron dyshomeostasis is an attractive proposition to investigate, as it is not specific to AD, but also observed in other neurodegenerative diseases. The close ties of iron to the pathological proteins (Robinson et al., In conclusion, heme/iron dyshomeostasis is an early and crucial event in AD pathophysiology, which warrants thorough investigation as a potential therapeutic target.https://ida.loni.usc.edu/pages/access/studyData.jsp.Publicly available datasets were analyzed in this study. This data can be found here: The studies involving human participants were reviewed and approved by As per ADNI protocols, all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. More details can be found at adni.loni.usc.edu. The patients/participants provided their written informed consent to participate in this study.AA contributed to the study concept, design and carried out the data analysis, and drafted the manuscript. MD helped revise the manuscript. P-WS contributed to data interpretation and revised the manuscript. All authors read and approved the final 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.