When listening with a cochlear implant through one ear and acoustically through the other, binaural benefits and spatial hearing abilities are generally poorer than in other bilaterally stimulated configurations. With the working hypothesis that binaural neurons require interaurally matched inputs, we review causes for mismatch, their perceptual consequences, and experimental methods for mismatch measurements. The focus is on the three primary interaural dimensions of latency, frequency, and level. Often, the mismatch is not constant, but rather highly stimulus-dependent. We report on mismatch compensation strategies, taking into consideration the specific needs of the respective patient groups. Practical challenges typically faced by audiologists in the proposed fitting procedure are discussed. While improvement in certain areas  is definitely achievable, a more comprehensive mismatch compensation is a very ambitious endeavor. Even in the hypothetical ideal fitting case, performance is not expected to exceed that of a good bilateral cochlear implant user. Among patients with asymmetric hearing, the largest interaural mismatches exist with one cochlear implant (CI) and acoustic hearing in the other ear . The acoTwo primary benefits can arise from listening with two ears: (1) Spatial release from masking, and (2) azimuthal sound localization and the related spatial perception of a sound field. Both benefits are based on the directionally dependent interaural time differences (ITDs) and interaural level differences (ILDs). The ILDs arises from a frequency-depended head shadow and their primary benefit can be understood acoustically, i.e. without considering ear or brain mechanisms. Different ILDs for sounds from different directions often provide a better signal-to-noise ratio (SNR) at one of the two ears, translating into an appropriate masking release, called the head-shadow effect. At high frequencies ILDs can be as large as 20 dB, constituting a potent cue for deciding whether the sound source is on the left or on the right . The benThe degree to which asymmetrically impaired patients can still benefit from binaural processing in the brainstem arguably depends on the fitting of their devices. The three critical fitting dimensions are level, latency, and frequency . A mismatch in any one of these dimensions can obliterate the benefits of binaural processing. To make matters even more complex, the interaural difference in each dimension is frequency-band specific. For level - which is arguably the most important fitting dimension - it is not even clear what a “matched level” means and what the fitting goal should be. Furthermore, an ideal fitting for the level dimension must be more than simply adjusting an amplitude-scaling factor. Rather, the output amplitude has to be a function of baseline level, and, due to different compression and adaptation effects in the devices and in the auditory system, the optimal level mapping even depends on the short-term input history e.g., .Asymmetric hearing can disrupt binaural fusion. Normal-hearing and most symmetrically impaired listeners take binaural fusion for granted: A single sound source is perceived as a single object. Without fusion, there are two acoustic objects, one at each ear. Fusion is not binary; it can be partial and it can depend on the stimulus. The fundamental importance of fusion for binaural fitting is that it alters even how the experimenter has to ask the question and even the fitting goal. For example, in case of fusion, there is just a single percept and hence a single loudness. There is no left loudness and right loudness in this case, and no possibility for actual loudness balancing when both ears are stimulated simultaneously . ReverseGiven this complexity, it is not surprising that until now, bimodal and SSD-CI users are mostly fitted as if their contralateral hearing would not exist. Usually, only a coarse, broadband loudness matching is performed. In the context of research studies, compensation of mismatch has been addressed in all dimensions , but a cIn the following, we first review the sources for interaural mismatches and elucidate their relation to brainstem processing and subsequent perceptual consequences. Next, we list mismatch measurement techniques, and describe what exactly they measure, as well as their limitations, interdependencies, and efficiency. While these two sections have primarily a review character, the next two sections aim at providing a perspective for future directions: There we assume that we know the mismatch and then elaborate on strategies that are expected to reduce the interaural mismatches, while considering their side effects. The last section is an attempt to give a clinical outlook: What tools are going to be required, and which measurement and fitting parameters are expected to provide the best return on time investment, for each patient group? Each section contains a subsection for each of the three dimensions: latency, frequency, and level. The focus is on post-lingually deaf adults with no other medical issues.When speaking of interaural differences, this commonly refers to acoustic differences in the sound fields at the left and right outer ear. In the present context, however, where the left and right inner ear are stimulated by different modalities, we refer to an interaural mismatch as any type of left-right bias introduced by a hearing device and by the asymmetric auditory pathway. For a sound that is emitted frontally, i.e., with no acoustic differences between the two outer ears, the bias can refer either to a different left-right auditory nerve (AN) response, or to a perceptual lateralization bias.Sound processing along the auditory pathway requires a certain amount of time. In a normal-hearing listener, this processing latency is identical for the left and right ear. Assuming the same devices on both ears, the processing latencies for bilateral CI users should also be identical between ears, and they therefore will not suffer from latency mismatches. However, in SSD- and bimodal CI users, the early stage of the auditory pathway up to the inner ear is replaced by the electrical pathway of the CI system on one side. The most relevant peripheral contributions to the latency are HA processing and the inner ear on the acoustic side, and the CI processor on the electrical side .With acoustic hearing, the sound wave arriving at the outer ear needs about 74 µs to travel through the ear canal and up to 250 µs to cross the middle ear . In relaWhen listening with a HA, a processing latency is added to that of the auditory pathway. Depending on HA brand and type, processing latencies vary between 2 and 10 ms. Some devices have a constant latency, while others are frequency dependent . Higher In the case of SSD-CI users, and provided there is a comparable neural processing latency, the FIR filter-based CIs cause a fairly low overall latency mismatch between acoustic and electrical hearing, as the FIR latency is comparable with the delay of the inner ear  is not a direct correlate of loudness . UltimatDue to the asymmetric status in bimodal or SSD-CI users, the two ears can be expected to have a different number of AN fibers and/or a different tonotopic distribution of fibers. As such, a comparable stimulation leads to different compound responses, even if isolated fibers respond similarly. Secondly, acoustically stimulated AN fibers have very different sensitivities, resulting in dissimilar rate-level functions. Such a range of properties is not observed with electric stimulation , so thatDue to the hair-cell synapse, spike-rate adaptation on the acoustic side is stronger, as are adaptive gain-control mechanisms, especially via the efferent innervation of outer hair cells. This causes dynamically changing interaural response rate differences and differently steep level-growth functions resulting in interaural differences in loudness growth . Both HATo date, HAs often operate with frequency-channel-specific AGCs , whereasYet, another reason for response rate differences comes from general stimulation or excitation limitations. The impaired acoustically stimulated cochlea may not be sufficiently sensitive at the basal end, whereas electric stimulation may be limited near the apex or to avoid unintentional electrical excitation of the facial nerve . As a coThe long list above focused on differences manifested as different AN response rates. As noted at the beginning of this subsection, perception may not necessarily reflect these rates. Especially when the left and right stimulation fuse into a single object, we expect a very different situation than without fusion. Without fusion, we perceive two separate images and an interaural level mismatch may cause a loudness imbalance  and on vTo be able to compensate for the interaural mismatches and to facilitate an improvement of binaural benefits (see section “mismatch compensation and side effects”), one has to first find the proper measurement tools to determine the amount of the respective mismatch. In this section, we summarize the current possibilities to do so and point out their advantages and their limitations.To determine the interaural mismatch in latency between the acoustic and the electric ear, the processing time of each side has to be known. This can be achieved by determining the processing time of the auditory pathway including the hearing device, or by determining the processing time of each component separately, i.e., the hearing device latency and that of the auditory pathway in separate measurements. The prime measurement method for both possibilities is the measurement of the wave V latency via (e)ABRs. As long as the auditory pathway is responding to the stimulus, wave V is a robust peak that can be identified in the brainstem response even with electric stimulation, and therefore allows for a reliable prediction of the processing latency along the auditory pathway .The combined device and patient latencies of the acoustic-hearing side, can be measured in a free-field environment, e.g., using loudspeakers, or by HA via audio cable . The twoFor the electrically stimulated side the same question arises; whether to measure the whole system, or device- and neural latencies separately. In the case of a combined measurement, CI pulses can be emitted during the moment that the brain response is occurring. To avoid these pulse artefacts that are much larger than the brain response, a stimulus design that knows how the sound-processing strategy behaves is needed. The more direct way forward is to stimulate the CI electrodes directly and measure the neural latency in isolation. Pulse artefacts will still be present, but precede the response. Even if the artifact leaks into the response time window, it is fully deterministic in the case of direct stimulation and methods are available to subtract it e.g., .The CI device latency may not be known, but if latency mismatch compensation is going to be part of the bimodal fitting process, it can be expected to have been taken into account by the manufacturer-specific fitting software. In the meantime, CI device latency can also be determined by direct electric measurements at the CI electrodes . The pulOverall, wave V-based latency measurements are possible and informative. For the most important frequency-specific latencies, however, errors larger than 1 ms can often not be avoided. Additionally, clinics might not have access to (e)ABR settings to reliably measure wave V-based latencies. Taking these two points into account, average (e)ABR latencies, or estimations from an individualized simulation  offer a Different approaches have been tried to determine the specific interaural tonotopic mismatch. The different measurement techniques are psychoacoustic, image-based, or based on the BIC derived from auditory brainstem responses. In this section, we discuss the benefits and the limitations of these methods.The arguably fastest measurement technique is imaging. With the help of x-ray or computed-tomography (CT) images, the electrode position within the cochlea can be measured by estimating the insertion angles of the electrode contacts. Due to the tonotopic order of the cochlea, the insertion angle can be assigned to a specific center frequency using the Even without the availability of an image, the position of electrode contacts and its corresponding frequency can be estimated. Therefore, the surgical information about the insertion depth and technical information about electrode length and spacing between electrode contacts can be used to calculate the position within the cochlea . If avaiThe most commonly employed behavioral method to measure tonotopic mismatch is pitch matching . The acoMeasuring ITD sensitivity while varying the place of stimulation in one ear builds on the finding that a tonotopically matching stimulation results in maximum ITD sensitivity . In bilaAnother difficulty that arises with the transition from bilateral to bimodal is that latencies may no longer be matched, causing an extreme bias to any static ITD task. This problem can be reduced by first matching the latencies (see section “latency mismatch compensation” and “latency fitting”) or by using non-singular ITD values, such as a large range of different, fixed ITDs , or dynaLast, it is possible to determine the tonotopic alignment using the BIC derived from auditory brainstem responses . Most coVarious behavioral and evoked-response based measurements have been suggested to derive the interaural level mismatch in CI users . Most coIn clinical fitting, the loudness balancing or centralization task is normally done while listening with both ears to a relevant broadband signal such as speech or speech-shaped noise, and typically only for one signal level. For psychoacoustic research or for frequency-specific compensation, it is also possible to perform the task with single electrodes and a more narrowband acoustic signal. However, it might be necessary to measure and compensate for the interaural frequency mismatch first, as frequency ranges of the electrode-to-frequency allocation might shift during the compensation process (see section “frequency mismatch measurements” and “frequency mismatch compensation”). To match levels across the dynamic range, a direct measurement becomes very time consuming. Adaptive measurement techniques , or modeA very different approach is to use evoked, response-related measurements, allowing level estimates and balancing at specific stages of the auditory pathway, for example in the brainstem and midbrain by means of auditory brainstem response (ABR) amplitudes  or late The starting assumption is that the patient has a mismatch in latency, frequency, and level and that these mismatches even vary with many stimulus and device parameters; most of all they differ for each frequency band. When trying to compensate these three mismatched dimensions, one needs to be aware of interdependencies, e.g., a change in HA gain also changes the wave V latency, due to the level dependency of ABR latencies . AnotherAs discussed in section “latency mismatch measurements”, the goal is to match latencies between sound arrival and a certain neural biomarker, such as the ABR wave V. Latencies can only be matched by increasing the device latency on the side with the shorter latency. In practice, most devices do not allow for a latency adjustment, and if they do, it is a frequency independent delay. In this subsection, we describe how the latency would have to be adjusted in the optimal case and ignore the present device limitations. The type of adjustment depends primarily on the CI processing type (FFT- or FIR-based filter bank) and the acoustic processing . Compensation requirements resulting from the examples of latencies shown in In the case of SSD-CI users with FIR-based processing, the interaural latency mismatch is relatively small and only at high frequencies does the CI side have a slightly shorter latency . Thus, tIn bimodal CI users, the HA processing latency is an additional component to be considered for latency mismatch compensation. The easiest cases are bimodal CI users with FIR-based processing on the CI side and frequency independent processing in the HA. As for SSD-CI users, despite the mismatch in latency, the electric as well as the acoustic side have a similar frequency-response curve that can be compensated for with an additional constant delay on the CI side. For some manufacturers, a latency compensation using a constant delay is already included into their fitting software, e.g., MED-EL Maestro 9. If bimodal CI users would be provided with a HA with a frequency dependent latency , a frequency dependent delay on the CI side is ideal.In bimodal CI users with an FFT-based CI, the device latency is frequency independent but relatively large. Depending on the HA latency, it is possible that the latency on the acoustic ear is shorter than on the CI ear. In that case, a frequency-dependent compensation has to be performed at the HA, as the latency difference can be expected to increase with increasing frequency. Another complication for bimodal CI users might be patients with an open HA fitting. In addition to the processed signal, the direct sound path may play a role, especially at low frequencies  and withIn bimodal CI listeners, an acute compensation of the latency mismatch by adding a constant delay to the CI side was performed by In SSD-CI users, a compensation of the interaural frequency mismatch is not possible at the acoustic-hearing ear. Also in most mild- to moderately hearing-impaired patients it can be assumed that there is no frequency compression employed in the HA. Furthermore, in cases where the acoustic ear is the better ear, it would not be prudent to possibly distort the speech signal by doing compensations at the acoustic ear. Therefore, the compensation has to be implemented at the CI side. Normally, the frequency band delivered over the CI electrodes differs deliberately from the respective Greenwood frequency, to provide the complete frequency range (e.g. 150–8000 Hz) required for optimal speech intelligibility . HoweverAlso the usability of evolutionary algorithms to optimize the frequency fitting is currently investigated and shows promising results concerning speech outcome and sound quality, whereas specific binaural benefits are yet unknown .There are hints that bimodal CI users might be tolerant to small frequency mismatches and that for deep inserted electrodes, a compensation of the frequency dimension alone might not have a large impact on binaural benefits. With an average frequency mismatch of 0.15 octaves, Compensating the mismatch by shifting the frequency-to-electrode allocation, it is important to address deactivated electrodes or neural dead regions as well. It is well known that the existence of cochlear dead regions can constrain the benefit of combining acoustic with electric stimulation . In thesIf the latency of the filter bank of the CI speech processor is frequency dependent, an inter-dependence of latency matching and tonotopic matching has to be expected. This has already been observed in one bilateral CI user, where it was assumed that a tonotopic mismatch induced an otherwise absent latency mismatch .When conceiving a bimodal or any type of bilateral fitting, arguably the first thought is on adjusting the stimulation level. The most common fitting-goal description in this respect is “loudness balancing” . LoudnesOn the other hand, reducing an interaural level mismatch may not be a desired goal in the first place. Especially in the absence of binaural fusion, there is apparent value in optimizing each device by itself in an attempt to maximize speech intelligibility . In caseThe two most common practices are left- and right-loudness balancing e.g.,  and centIn cases where the patient has a binaurally fused percept, fitting towards a centralized perception should be favored (see section “level mismatch measurements”). If the degree of binaural fusion is unclear, a centralization setup is still possible and arguably ideal, because even CI users with partial fusion or without fusion will be able, with the alternative instruction to match loudness instead of centering the sound image, to find the level at which both percepts are equally dominant or equally loud.Due to different loudness growth between electric and acoustic hearing, the relative levels necessary to achieve a balance dependents greatly on the absolute level , and canAdditionally, dynamic aspects of the processing in the devices and in the auditory system may disrupt the ILD cues. Matching the parameters of the AGC, including the time constants and the knee points, can decrease the mismatch at least in cases of severely impaired hearing at the acoustic ear . ContrarOverall, level balancing appears to be one of the most important and one of the most difficult fitting aspects for bimodal CI. Much has been done, and much more can be done in the future.In the previous sections, we discussed the causes of the interaural mismatches , mismatch measurement techniques (section “mismatch measurement techniques”), and compensation strategies (section “mismatch compensation and side effects”) for each of the three dimensions level, latency, and frequency. In section “mismatch compensation and side effects”, we noted how inter-dependent the three dimensions are, and that a large gap remains between knowing these strategies and having a comprehensive and practicable fitting protocol. The goal of the present section is to describe the various aspects of this knowledge gap and to discuss some paths that researchers and audiologists follow or may follow in the future to jointly work towards a clinically feasible bimodal fitting protocol. One focus is to work out the consequences of the interdependencies for the measurement order and compensation order. To limit the complexity and number of different cases, we primarily consider the case that unilaterally optimal fittings of both HA and CI exist and that the patients’ acoustic hearing ear is considered the “better ear”, e.g., with respect to speech understanding. We make the simplifying assumption that in such a case, level and frequency mismatches are best compensated for by altering the CI parameter settings such that the better ear performance is not compromised.In the same vein as in the previous sections, an ideal facility is envisaged. Practical limitations, such as the availability of imaging equipment or staff expertise to fit both HA and CI, play a central role in how a clinic organizes the fitting routine. Here, we will generally assume a best-case scenario, but are aware of inevitable constraints, such as the available time per fitting session, or patients’ ability and willingness to perform extended listening tasks. We will also point out where the present reality differs or is expected to differ from the best-case scenario. Just as above, this section does not address pediatric fitting or the fitting of patients with severe hearing loss in the acoustically stimulated ear.The best-case scenario for the fitting procedure of either newly or long-term implanted patients is based on the following assumptions: (1) The electric and acoustic latency up to wave V (including the device latency) of a bimodal or SSD-CI user is already known or confidently estimated from known device latencies (see section “frequency mismatch measurements”). (2) A CT image of the inserted electrode array is available, no electrodes are deactivated and no dead regions are present. (3) It is possible to flexibly change CI stimulation levels and frequency allocation and to increase processing latency at either device in a frequency specific manner (The latter cannot yet be expected in practice). Apart from optimistically assuming that this technology is readily available when these lines are read, we follow the previous sections with typical present-day devices and technology in mind, such as the clinically available speech-coding strategies.At the end of the fitting procedure, the optimal outcome would be that the spatial hearing performance of bimodal users reaches that of their bilateral CI peers . The maiLatency compensation is an ideal starting point, because it is less affected by the other stimulation parameters or mismatch factors. Evidently, the device on the side with a shorter compound latency should be delayed in a frequency-specific manner see . In the Following the compensation of the latency mismatch, the next step is to reduce the frequency mismatch  by adjusApart from our best-case scenario, there might be cases in which no postoperative image of the inserted electrode is available. With pitch matching having several disadvantages and determining the BIC facing several challenges (see section “frequency mismatch measurements”), the measurement of ITD sensitivity along the electrode array might be a good alternative, as well as estimating the corresponding frequency using surgical and technical information (see section “frequency mismatch measurements”). The latter might be an option especially for CI users, that struggle with sensitivity towards ITD. Additionally, it should be noted, that although not all clinics perform post-operative CT scans, in most cases at least a post-operative x-ray image is part of the clinical routine. Therefore, the x-ray can be a good compromise to perform image-based fitting if a good quality of the image can be provided.Newly implanted CI users do not initially tolerate high stimulation levels, and their sensitivity to level changes considerably over the first few weeks. It is unlikely that a time-consuming precision adjustment is in the interest of either the clinician or the patient, given the fitting's short expected life span. Only after completing the acclimatization phase does it make sense to compensate the level mismatch between the acoustic and the electric ear, and the existing fitting can be used as a starting point. We expect that for the most part, the adjustment will be performed broadband. An example approach is illustrated in As a first step, we propose testing whether a frontally presented, high-level stimulus is perceived as too loud. As little is known about binaural loudness in bimodal and SSD-CI users, the possibility exists that the bilateral presentation is far too loud, despite each unilateral stimulation in isolation being acceptably loud . This caFurther fine-tuning of the level parameters is possible by using a more complex approach, with a frequency-specific narrowband stimulation. This approach allows for a more accurate adjustment of the level parameters. However, to reach the necessary accuracy with frequency-specific measurement techniques, long measurement times are necessary, which means that such methods are most likely not time-efficient enough for a clinical setup. Some novel approaches to overcome this inconvenience while preserving the needed accuracy - e.g., improving the efficiency of categorical loudness scaling, are currently being examined e.g., . It shouAlthough we have suggested the fitting order: 1) latency, 2) frequency, 3) level for our best-case scenario, it may be necessary to alternate between the three fitting dimensions or to iterate through the process for a second time. This is due to the interdependencies between the dimensions. Latency, for instance, which we have argued to be least dependent on the other parameters, nevertheless depends on level in acoustic stimulation, but much less so in electric stimulation . SimilarIn the previous sections (“latency fitting” to “interdependencies”), we assumed a best-case scenario. As the name suggests, this is clearly not a “one fits it all” guideline, but is rather expected to have various practical limitations.First, the control of one of the devices may be limited either by manufacturer constraints or by staff-specific limitations. An example for the former is that not all CIs allow for a completely custom frequency allocation. An example for the latter is that HA and CI fitting is often performed in a sequential manner by two different professionals. Similarly, whereas each device is typically fitted within its own framework, a combined CI and HA fitting software is arguably the best approach, but only available for a few combinations of partner-brand devices e.g., .The theory behind bimodal fitting laid out in this and other articles is so complex that even dedicated researchers may not always be able to fully grasp the complex interplay. Concentration on some essential components will be inevitable and typical. At this stage, the suggestions presented here primarily address early adopters, such as research audiologists in large centers. Knowledge translation to the clinical routine is then the second step, but needs to be considered early on e.g., . ParticuAfter setting all parameters, it is important to verify if the fitting improves hearing in tests that reflect real-life. For newly implanted patients a comparison towards the preoperative results can be achieved. However, it rather displays the success of a fitting compared to no CI rather than the actual success of the compensation itself. Nevertheless, it might give some insights, if further optimization of the fitting might be necessary and serve as a baseline for longitudinal improvement or future fittings. In contrast, for long-term CI users who may have received their first “binaurally optimized fitting” to compensate for their mismatches, a direct comparison before and after the mismatch compensation is possible and allows a judgement about successful mismatch reduction. To allow comparable outcome measurements among different centers, A reduction of interaural mismatch in frequency and latency improves binaural fusion. Without binaural fusion the two ears act as two almost independent receivers. With binaural fusion we expect better localization and possibly improved masking release but we have to revisit some concepts such as loudness balancing and anticipate a more involved fitting process.The three dimensions level, latency, and frequency are interdependent.A mismatch in one dimension can obliterate the benefits of matching in other dimensions.Level balancing is not always expected to be possible such that the patient perceives all frontal sources from the front.This sobering summary is part of the reason why an elaborate bimodal fitting protocol is far from clinical routine. Binaural fusion is critical in formulating the fitting goal, but often not considered. With improving device technology, such as adjustable latency, or a wireless information exchange, and with more bimodal patients with good acoustic hearing, the demand for a smart fitting strategy will increase. Fitting tools are also improving, most notably CT-based imaging, but the task is not expected to get much easier.The complexity of fitting SSD- and bimodal CI patients is reflected in the length of the present text. Four examples of conclusions distilled from the literature are:


The present study was designed to evaluate the dynamic expression of lncRNA NORAD in fracture healing of patients with brittle fractures and explore the function and mechanism of NORAD in regulating osteoblastic proliferation, differentiation, and apoptosis. The expression level of NORAD was detected by quantitative real-time PCR. The proliferation, differentiation, and apoptosis of osteoblasts were analyzed by MTT assay, ELISA, and flow cytometry. Luciferase report analysis was used to confirm the interaction between NORAD and its target ceRNA miR-26a. This study showed no significant differences in serum NORAD expression on the 7th day during fracture healing in patients, but increased expression of NORAD was certified on the 14, 21, and 28 days after fixation. Overexpression of NORAD promoted the proliferation and differentiation of osteoblasts and suppressed the apoptosis of osteoblasts. miR-26a proved to be the target gene of NORAD and was inhibited by overexpression of NORAD in osteoblasts. The enhanced expression of miR-26a was negatively linked to the lessened expression of NORAD. NORAD could accelerate the proliferation and differentiation of osteoblasts and inhibit apoptosis, thereby promoting fracture healing. Osteoporosis (OP) is one kind of endocrine disorder characterized by decreased bone mineral density, damaged bone microstructures, and osteopenia, which often occurs in the elderly . BrittlelncRNAs are endogenous noncoding RNA molecules that affect numerous biological activities. lncRNAs are involved in the management of mRNAs' expression, influencing different organic processes, like bone metabolism . lncRNA In this research, NORAD was inferred to be correlated with fracture healing. Patients with brittle fractures were selected to examine the expression of NORAD. The expression of NORAD was tested during the progression of fracture healing. Furthermore, the impacts of NORAD on proliferation, differentiation, and apoptosis of mouse osteoblast cell line (MC3T3-E1) were analyzed.A total of 87 patients with brittle fractures treated in BinZhou Medical University Hospital were recruited in this study, including 47 patients with hand fractures and 40 patients with intraarticular fractures. The patient's group included 42 males and 45 females with an average age of 65.19 ± 8.71 years old. In addition, 43 healthy participants were recruited as healthy controls. There were 21 males and 19 females in the control group with a mean age of 64.40 ± 9.18 years old. Exclusion criteria for patients were as follows: (1) younger than 18 years old, (2) malignant tumor, (3) inflammatory arthritis, (4) heart failure, kidney failure, and (5) autoimmune diseases. An approvement by the ethics committee of BinZhou Medical University Hospital was sought, and an informed consent form was signed by each participant. Blood samples of 5 ml were collected from each patient within 24 hours after fractures, 7 days, 14 days, 21 days, and 28 days after standardized fixed treatment. The blood sample was allowed to stand for more than half an hour and centrifuged at 3500-4000 rpm for 5-10 minutes to get the serum. Serum specimens were deposited at -80°C for later use.All MC3T3-E1 cells were purchased from Shanghai Cell Bank, Chinese Academy of Sciences, and cultured in the alpha-minimum essential medium  containing 1% penicillin-streptomycin  and 10% fetal bovine serum.The oligonucleotide sequences of NORAD from GenePharma  were cloned into pcDNA 3.1 vectors to build overexpression carriers. Besides, the si-NORAD from Gene Pharma was used to interfere with normal NORAD expression. According to the manufacturer's instructions, instantaneous transfection of synthetic molecules was carried out using Lipofectamine 3000 reagent . All following experiments were conducted after two-day incubation.-∆∆CT method. U6 and β-actin were used as internal reference genes.TRIzol LS reagent  was added to serum and cells, and total RNA was isolated and extracted. For the NORAD, the first-strand cDNA synthesis mixture of Hifair  was used to synthesize the cDNA. For miR-26a, the total RNA was synthesized into cDNA by using an miRNA strand 1 cDNA synthesis kit . Then, an miRNA qPCR kit  was used to amplify the cDNA sample of miR-26a by qRT-PCR. The qRT-PCR was used to amplify cDNA samples of NORAD with qPCR premix . The relative expression was calculated by the 24 cells were spread in 96-well plates, then 20 μl MTT reagent was added and incubated at 37°C for 4 hours. Following that, 150 μl Formanzan reagent was added. After being incubated for 4 hours, the absorbance of each well was measured at 570 nm.At 0, 24, 48, and 72 hours after cell treatment, the proliferation of MC3T3-E1 was evaluated by MTT . About 5 × 102 at 37°C. Bone morphogenetic protein 2 (BMP2) was used for the differentiation induction of MC3T3-E1 cells. MC3T3-E1 cells were inoculated into a 6-well plate with a density of 106 cells per well and cultured in the medium containing 200 ng/ml BMP2 for 14 days, and the medium was refreshed every 2 days. The differentiation of MC3T3-E1 cells was reflected by the levels of alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), collagen 1, and osteopontin (OPN). ELISA kits from Elabscience Biotechnology  were utilized for the identification of cellular differentiation after cells being differentiated for 14 days.Cells were cultured in a humid environment containing 5% COμl Annexin V-FITC binding solution was added to resuspend the cells. A total of 5 μl annexin V-FITC and 10 μl propidium iodide (PI) were added to each well, and the plates were incubated in the dark for 10-20 minutes at room temperature. The fluorescence intensity was detected by flow cytometry. The kit used in this experiment was the Annexin V-FITC apoptosis detection kit .Cells were incubated for 24 hours and digested with trypsin free of EDTA. The cells were collected and washed twice with phosphate-buffered saline (PBS). After centrifugation for 5 min, the supernatant was discarded, and 195 μl of the sample was mixed with 100 μl of luciferase detection reagent, and the relative luciferase activity was measured by a chemiluminescence instrument.The target relationship between miR-26a and NORAD was detected by a double luciferase reporter gene detection kit . Before detecting the relationship between miR-26a and NORAD, the pmirGLO vector was transfected with wild-type (WT) or mutant (MUT) 3′-UTR of NORAD to build WT-NORAD or MUT-NORAD vectors. After cloning, the two plasmids were transfected into cells together with miR-mimic, miR-inhibitor, mimic-NC, or inhibitor-NC, respectively. After transfection, these cells were lysed. Then, 100 t-test and one-way ANOVA were used for comparison between groups. P < 0.05 is used to demonstrate the statistically significant difference.GraphPad 9 was used to analyze the data. Student's P < 0.001). The expression of NORAD in male patients group and female patients group exhibited a similar tendency to that in total patients (P < 0.001). However, there was no difference in NORAD expression between female and male patients in control group, hand fracture group, and intra-articular fracture group (P > 0.05).The expression level of serum NORAD in patients with hand fracture and the intra-articular fracture was detected by qRT-PCR. The expression of NORAD in patients with hand fracture and the intra-articular fracture was both decreased compared with control individuals (P < 0.01). The same result was certified in the healing phase of patients with intra-articular fractures. On the 14th, 21st, and 28th days, the expression level of serum NORAD in patients with intra-articular fracture was significantly higher than those on the 1st day (P < 0.05). The results indicated that the imbalance of NORAD expression might change with the stage of fracture healing.As seen from P < 0.001).In addition, to investigate the effect of NORAD on fracture healing, NORAD was overexpressed or interfered with artificially. The level of NORAD was increased in the pCDNA3.1-NORAD group, and inhibited in the si-NORAD group, confirming that NORAD could be regulated by pCDNA3.1-NORAD and si-NORAD in vitro (P < 0.001). Differentiation of osteoblasts is one of the important progression of bone formation, thus, we further confirmed whether NORAD was involved in osteogenic differentiation. We upregulated or downregulated NORAD in differentiated MC3T3-E1 cells and then detected the levels of osteogenic differentiation markers, including ALP, Runx2, Collagen I, and OPN. As shown in P < 0.01). In addition, overexpression of NORAD prevented cell apoptosis, while reducing the expression level of NORAD led to the augment of cell apoptosis (P < 0.001).An increase in MC3T3-E1's proliferation was found in the NORAD overexpression group, while interference of NORAD inhibited the cell proliferation , while the luciferase activity in NORAD-MUT group was unchanged (P > 0.05). Increasing NORAD suppressed miR-26a levels, whereas inhibiting NORAD exerted the opposite function (P < 0.001).After cotransfection of the miR-26a mimics or miR-26a inhibitors with NORAD-WT 3′UTR, luciferase activity was significantly inhibited or increased (P < 0.001). In different gender groups, the expression of miR-26a was elevated in patients with intra-articular fracture and hand fracture (P < 0.001), while no discrepancy was found on miR-26a levels due to the gender difference (P < 0.05). Additionally, the opposite tendency of NORAD expression and miR-26a expression was found in inpatients with hand fracture  and intra-articular fracture . In both hand fracture and intra-articular fracture, the expression of miR-26a was lessened with the fracture healing . All the above results further confirmed that miR-26a directly targets NORAD in fracture.The ascended expression of miR-26a was identified in patients with hand fracture and intra-articular fracture (Fractures are common traumatic damages, and nonunion and delayed union of fractures impose a severe economic and mental burden on patients . The incAccumulating evidence prove that lncRNAs may broadly contribute to tuning the expression pattern of specific downstream genes . SeveralMost lncRNAs are pertinent to the regulation of miRNAs, and lncRNAs can reduce miRNAs' regulatory effect on mRNAs by acting as miRNA sponges . lncRNA Taken together, the expression of NORAD was diminished in patients with fractures and gradually fractured during the fracture healing. Enforced NORAD expression exerted a beneficial function on fracture recovery by promoting osteoblastic proliferation and differentiation and inhibiting apoptosis of MC3T3-E1 cells. MiR-26a is a ceRNA of NORAD in fracture. Fracture was conducive to the increment of miR-26a. A reverse trend of miR-26a expression and NORAD expression was pinpointed in this research.


Assessment of hepatic venous pressure gradient (HVPG) is the most reliable, though invasive method for evaluation of portal hypertension. Non-invasive, elastography-based techniques are well established in diagnosis, but not in monitoring of portal hypertension. The aim of our prospective study was to determine the value of acoustic radiation force impulse (ARFI) elastography technique of the liver and spleen in diagnosis and monitoring of portal hypertension.  We prospectively assessed portal hypertension by HVPG and corresponding elastography of the liver and spleen in 31 patients with liver cirrhosis and an indication for primary prophylaxis by non-cardio selective beta-blockers. Investigations were performed at baseline and a follow-up visit after 6-8 weeks. To address the known large variability of values for spleen elastography, well-defined corresponding areas in the spleen were used for baseline and follow-up elastography. Sensitivity, specificity, and AUC-ROC values for both spleen and liver elastography monitoring of portal hypertension were calculated.  Liver but not spleen elastography significantly correlated with HVPG results and was suitable for initial evaluation of portal hypertension. However, changes in HVPG results did not show any correlation with alterations of ARFI values from baseline to follow-up visits both for liver and spleen elastography. Spleen stiffness results were not homogeneous across the whole organ differing significantly between the upper, hilar, and bottom placed investigation areas. NCT03315767. In this prospective study ARFI-based assessment of liver elastography showed itself suitable for initial assessment but not for monitoring of portal hypertension. Spleen elastography was not appropriate for both, evaluation and monitoring of portal hypertension. A possible explanation for this new data that are in some contrast to previously published results is the degree of portal hypertension in our study, a comparatively short follow-up period, and well-defined investigation areas for spleen elastography in repetitive ARFI investigations. This trial is registered with  Chronic liver diseases result in structural alterations in the liver with increasing fibrosis ultimately resulting in cirrhosis. Dependent on the kind of hepatopathy fibrosis can progress fast  or it can be a slowly progressive disease  , 2. The Hepatic venous measure gradient (HVPG) is calculated as the difference between the pressure in the portal vein and the pressure in the liver vein. Results of a HVPG investigation being >5 mmHg define portal hypertension and being >10 mmHg are associated with a clinically significant portal pressure, respectively . MeasureEvaluation of portal hypertension by assessment of blood results was performed in a considerable number of studies: a combination of albumin/ALT/INR score completed by clinical signs of advanced liver disease (spider naevi) results in excellent sensitivity for portal hypertension . HoweverNCT03315767). The trial conformed to the declaration of Helsinki [The trial protocol was approved by the local ethics committee , intolerance to NSBB treatment (n =2), or trial investigations not completed (n =10), 31 patients were enclosed in the final analysis with baseline and follow-up visits completed. The flowchart for enrollment process and schedule of study investigations is presented in 92 consecutive patients with clinical signs of portal hypertension and advanced chronic liver disease were screened for participation in this study in the University Hospital Ulm, Department of Gastroenterology, Nephrology, and Endocrinology  between 10/2017 and 03/2019. Considering inclusion and exclusion criteria, 47 patients with indication for primary prophylaxis by NSBB medication were offered to participate in the study with HVPG-based response monitoring scheduled. Due to refusal to sign informed consent  or metastases in the liver, intolerance to NSBB medication due to special clinical conditions , and renal insufficiency.Baseline investigations comprised an assessment of portal hypertension by HVPG, evaluation of spleen and liver elastography , and laboratory results, respectively. The NSBB medication was started with carvedilol at initial dose of 3,125 mg twice a day. The initial monitoring of side effects due to carvedilol and dosage adjustment was performed while patients were hospitalized and continued in the visits in our outpatient clinic. Frequency of the first clinical follow-up visits was scheduled dependent on the tolerance of the new medication and clinical conditions of the respective patient up to twice a week if necessary. In the outpatient follow-up visits, measurements of heart rate and blood pressure were regularly performed. Patients were told to perform further heart rate and blood pressure controls at home and inform the attending physician about the results for dosage adjustment. The dose for carvedilol was adjusted over the following 2 weeks until the highest tolerated medication was reached . The mean dosage for carvedilol used in our trial was 13.25 mg/d (6.25–25 mg/d). The follow-up investigations (HVPG and ARFI) were scheduled 6-8 weeks later.HVPG (hepatic venous pressure gradient)The investigations were performed as described previously and will be briefly discussed in the following section:(ii) Liver and spleen elastography (ARFI technique)The HVPG investigations were performed by experienced gastroenterologists/hepatologists (>100 previous HVPG investigations). The procedures were done according to established standards . Brieflyth or 7th intercostal space, and it was ensured there were no major vessels or (liver) bile ducts in the B-scan in the area examined. All the recordings were performed in mid-respiratory position. The examination protocol required a measurement depth of at least 2 cm below the liver capsule or at least 5 cm from skin level. ARFI results were measured ten times in the liver and a mean value for each patient was calculated. For spleen elastography, 5 measurements were performed for the upper, the hilar, and the bottom third of the spleen, respectively, and a mean value was calculated for all the measurements in the spleen. In addition, mean values for the upper, the hilar, and the bottom third of every investigated spleen were calculated separately. For ARFI-based evaluation of the upper third of the spleen, a region of interest was mandatory set in a depth ≥5 cm; for the hilar third of the spleen, ARFI was performed in 5 cm depth and for the lower third ≤5 cm, respectively.All the elastography procedures were performed by physicians with specialization in ultrasound investigation and with experience in >100 prior elastography investigations (ARFI) of the liver and spleen. Ultrasound examiners were blinded regarding the results of the corresponding HVPG investigation. The evaluation of liver and spleen stiffness was performed within 24 hours of completion of the HVPG. A Siemens Acuson S3000  with a 6C1 convex transducer (1.0-6.0 MHz) was used for all ARFI examinations. The probe was placed in the 6t-test was used for values with normal distribution (the Shapiro-Wilk test performed). Fisher's exact test was used for determination of possible association of categorical variables. A binary logistic regression analysis was used to identify factors significantly associated with the response in a HVPG measurement. AUC-ROC and 95% CI were calculated for changes in liver and spleen elastography (continuous variable). In all the calculations, p-value <0.05 was defined to be significant. All the statistical analyses were performed using R Statistical Software  for Windows.Continuous variables were expressed as median and minimum–maximum values; categorical variables were expressed as numbers (and percentages). For the correlation between HVPG and elastography results before and after initiation of NSBB therapy, the Pearson correlation coefficient was calculated. The two-sided n =14). The median Model for End-Stage Liver Disease (MELD) score at baseline was 12.0 pts (9.0–15.5 pts.). 17 patients (55%) had Child A and 14 patients (45%) had Child B cirrhosis.31 NSBB therapy-naive patients with advanced chronic liver disease (ACLD), a HVPG-diagnosed portal hypertension and indication for primary prophylaxis, were included in this trial. The baseline characteristics of the patients are presented in p =0.03; p =0.009) at the follow-up visit (Baseline HVPG examinations showed a median gradient of 18 mmHg (10–27 mmHg). In 14 (45%) patients, a treatment response could be detected in the follow-up HVPG investigation, and 17 (55%) were NSBB non-responder. In patients with response to NSBB therapy, the median HVPG value significantly dropped from 20 (10-27) mmHg at baseline to 15.5 (2–20) mmHg  m/s . In patiThe baseline results for SSM of the whole spleen showed a median value 3.00 m/s (2.27–3.59). Neither patients with response nor patients with non-response to NSBB medication showed any significant changes in spleen stiffness evaluation: 2.96 (2.27–3.34) m/s (baseline) vs. 3.02 (2.33–3.22) m/s (follow-up) in the responder group ; 3.23 (2.38–3.59) m/s (baseline) vs. 3.03 (2.41–3.55) m/s (follow-up visit) in the non-responder group  .p = 0.00007 and p = 0.00008, respectively). The ARFI results assessed in the hilar section (2.98 m/s) and the SSM results of the whole spleen (3.00 m/s) did not differ significantly (p = 0.74) (Previously published results demonstrated a variability of stiffness results depending on the location of ARFI investigation in the spleen . Therefo = 0.74) .r = 0.82, p = 0.0003) at baseline visit between HVPG values and LSM results. This correlation was also detectable follow-up . However, there was only a weak, non-significant correlation between the changes in HVPG and LSM values from baseline to follow-up . Δ-SSM and Δ-HVPG results between the baseline and the follow-up visits did not show any correlation either  (ARFI investigations of the whole spleen did not show any significant correlation with the HVPG results both at baseline and at the follow-up examination ( =0.114) .Δ-HVPG results . The AUC-ROC result for delta-SSM was comparably low (42.23%) .2) and obesity condition .A binary logistic regression analysis of patients' data did not reveal any of the parameters being associated with HVPG-defined response. Especially, elastography values of the liver and spleen were not significantly associated with HVPG response . InteresThe results of our investigations showed that a non-invasive approach based on ARFI elastography does not accurately mirror HVPG-evaluated changes in portal hypertension in response to NSBB treatment. Liver elastography was able to detect portal hypertension and showed a strong correlation with measured HVPG values at baseline. However, in the follow-up investigation, delta-LSM was not able to allow a diagnosis of a successful response to initiated NSBB therapy compared with HVPG as gold standard. Furthermore, ARFI elastography of the spleen, performed in well-defined areas of investigation, showed only a poor performance for the diagnosis of significant portal hypertension as well as for the evaluation of the response status after NSBB treatment. In summary, stiffness assessment of both the liver and spleen by ARFI elastography was not able to reliably determine the response status to initiated NSBB therapy.Δ-HVPG and Δ-LSM with only little changes detectable, consistent with the result of other studies [Δ-HVPG results. Even more crucial appears the choice of well-defined anatomic areas for spleen ARFI investigation to perform repetitive measurements. Due to this issue, we defined three distinct positions for performance of repetitive elastography investigations in the most accurate and reproducible way. Using this approach, we were not able to see a positive significant correlation between Δ-HVPG and Δ-SSM. Furthermore, SSM evaluated by ARFI technique on standardized positions was not able to deliver a reliable detection of portal hypertension. This result was also obtained in some previous studies: Procopet et al. [Assessment of portal hypertension has attracted growing attention in the last decades due to its increasing prognostic and therapeutic clinical impact. Several studies demonstrated a strong correlation between the incidence of liver cirrhosis related complications, like variceal bleeding, hepatocellular carcinoma, ascites decompensation, and the level of portal hypertension , 27. Fur studies , 34. Fur studies : accordi studies , shear w studies , and aco studies . In the  studies , delta-S studies . The rest et al.  describet et al. . Furthert et al. –41. ThisThe time for the follow-up visit is also an important topic. Chronic HVPG response was evaluated in several studies with a follow-up interval for the repeated HVPG investigations >3 months after the baseline evaluation . HoweverThe basic characteristics of patients enrolled in our study do not show any significant differences compared with trials regarding treatment or prophylaxis of complications due to portal hypertension and advanced liver disease , 47. In Our study has some limitations: this single-center study was performed in a rather small number of patients with an accordingly limited number of investigators for both HVPG and ARFI evaluation. However, only few prospective studies exist for monitoring of portal hypertension by elastography techniques in correlation with HVPG results, that were performed with even a smaller number of patients compared with our trial . Due to In a prospective setting, our study demonstrates a good correlation between liver stiffness and HVPG values; hence, ARFI-based assessment of liver stiffness showed itself suitable for initial assessment of portal hypertension. However, the results of our study do not support a good correlation between spleen stiffness and HVPG values in advanced portal hypertension. Furthermore, we could not confirm a sensible role of spleen and/or liver elastography for the non-invasive evaluation of response to NSBB for a short follow-up period in patients with relevant portal hypertension. The interpretation of spleen ARFI results for evaluation of portal hypertension should be done cautiously for the spleen stiffness values are strongly dependent on the area of spleen investigated. Furthermore, high BMI and obesity seem to be limiting factors for a reliable repeated evaluation of liver and spleen stiffness for the surveillance of portal hypertension. Thus, HVPG remains the gold standard examination in this situation.


Once harnessing these concepts, we reach a juncture of an in-between space; entry points of nonidentity (sameness) and points where multiple effects of difference are created. Key discussion topics include: discourse, language and interpretations of literacy; in/tangibility of literacy; capturing literacy; literacy as a process or a product; connotations of the terms literate and illiterate; neoliberalism and literacy and finally literacy as learning. We believe that when understood as the productive and meaningful interaction with/in/through the world, literacy is still the appropriate term within the context of physical literacy. Our discussion leads us to conclude that as embodied individuals, physical literacy is often the literacy through which other literacies have to pass. Through physical activity individuals can not only nurture their own physical literacy but also contribute toward a global or holistic literacy that helps us navigate, connect and make sense of ourselves, others and the world around us. However, the paper acknowledges that this meaning is not always grasped with the historical understanding of literacy as well as it's translations into other languages presenting challenges in articulating the intended use, meaning and connotations of the contemporary understanding of physical literacy.The concept of physical literacy is continuing to gain traction internationally. This increasing interest has also given rise to concerns about the use, interpretation and meaning of the term “literacy” within the context of physical literacy. This paper explores the development of the terms literate, illiterate, literacy, and illiteracy identifying their historical origin and contemporary meaning. This provides the backdrop to explore the use of the term literacy within the context of physical literacy. In the final part of this introductory section the recent popularity of the literacies movement is explored. Our discussion identifies key intersections and areas of tension associated with the use, interpretation and meaning of literacy in the context of physical literacy. We adopt Whitehead's philosophy of physical literacy and discussion is informed further by Derrida's notion of  Education for All: Literacy for Life [United Nations Educational, Scientific and Cultural Organisation (UNESCO), In 2005, the United Nations Educational, Scientific and Cultural Organisation's (UNESCO) released participate fully in their community and wider society.” Being literate includes the use of critical and creative thinking skills and/or processes, conveying information through various forms of communication and applying knowledge and skills to make connections within and between various contexts [United Nations Educational, Scientific and Cultural Organisation (UNESCO), Literacy is typically viewed as falling somewhere on a continuum ranging from a set of skills to a basis for rational and ethical action  of literacy and second understanding(s) in the context of physical literacy. The contribution this paper makes is to explore historical and contemporary uses of the term “literacy” with the view to embedding a richer notion of it before it allies with “physical.” We identify and discuss key intersections and areas of tension which have emerged in relation to literacy as applied to physical literacy. Whilst providing insight into the intersections as we identify them, we draw from Derrida's  notion o Barad's  challeng Barad's  philosopliteratus/litteratus meaning “educated, learned, who knows the letters.” Its origin can be traced back to the early 15th century (15c.), with descriptions including “educated, instructed or having knowledge of letters.” By the late 18c. the term was frequently associated with being “acquainted with literature” and by 1894 also used as a noun, to mean “one who can read and write.” Literacy derives from literate, and also emerged in the late 18c. being described in 1883 as the “ability to read and write.” Similarly, the origins of “illiterate” can be traced back to the early 15c. with its meaning described as being “uneducated, unable to read and write.” Illiterate comes from the Latin illiteratus meaning “unlearned, unlettered, ignorant; without culture, inelegant.” The origin of Illiteracy can be traced back to the mid 16c. deriving from illiterate to mean the “inability to read and write.”The Online Etymology Dictionary  cites liThe use of these terms has naturally evolved and changed over time. Historically, literacy has predominantly been used in relation to English literacy, language and the ability to read and write. Prior to the late 20th century there were very few uses of the term “literacy” beyond its reference to the skills of reading and writing. However, in the last 20 years a range of areas have adopted the suffix of literacy. The number of areas adopting the term is now probably over two dozen, ranging from emotional literacy to food literacy and from environmental literacy to political literacy. In a brief literature search the following literacies were identified: Mathematical , Sum et al.  cited in Cairney et al.  describeWhitehead  describeThere was a significant period of time between Whitehead's doctoral thesis 1987) and her inaugural paper entitled “The Concept of Physical Literacy” introduction of the construct physical literacy interpretations of physical literacy as people “make sense” of the concept, in their own contexts. Currently, the International Physical Literacy Association [International Physical Literacy Association (IPLA), While it is of note that physical literacy is in no way a recently developed concept, it is worthy of mention that few of the earlier references appear to be related or founded on any clearly articulated philosophical principles that Whitehead's contemporary concept has at its foundation Bailey, . Physicacontroversies”   has usedBeyond its conventional concept as a set of reading, writing and counting skills, literacy is now understood as a means of identification, understanding, interpretation, creation, and communication in an increasingly digital, text-mediated, information-rich and fast-changing world.At policy, development agenda and implementation levels, there remains the central drive to improve “basic reading and writing skills” [Global Alliance for Literacy, United Nations Educational, Scientific and Cultural Organisation (UNESCO), Building from the challenges of multiple interpretations of literacy is the notion or idea that literacy is not a fully tangible concept. From the earliest scholars writing on the history of literacy to current literature, the term literacy remains untethered, a ‘will o' the wisp' character. Its application, ironically through literature, to an increasing number of society's practices reflects a simultaneous increase beyond text-based associations. “Multiliteracies” or “alternative literacies” are terms discussed by Provenzo et al. , drawingThe variety of methods, intangible as some might appear, need not be hierarchical capacities for interaction or communication. For instance, shorthand methods of producing the written word do not necessarily preclude conventional literacy development , neither captured nor absolute. Examples of this can be found when cooking, playing a musical instrument or sending a ball and moving to space. Therefore, physical↔literacy is a living library that documents thoughts, interactions, and musings through chaptered experiences  or reinforcing stereotypical desirable moving bodies, which can be elitist and exclusionary in nature. Instead, we advocate that the deliberate act of reflection, by the participant independently or supported by others, is the valuable learning taking place, and helps ensure that inclusion is centrally informing an embracing of wide-ranging embodied lived experiences.elitist” construct  literacy, running against the prevalence of calls for definition clarity. In refining the concept of literacy, it becomes restricted, and tightening in this way ensures the term is exclusive Sims, . UnderstPhysical literacy is currently discussed in academic literature, as well as being embraced in policy, such as Canada's Physical Literacy Consensus Statement  or some Literacy as interaction and communication represents the human individual as forever searching for meaning, resolving dissonance, finding balance and harmony. It is an expression of intentionality—the endless urge to relate, to affirm our being, to make sense of the world and ourselves . Snowboarders completing a half-pipe event at the Olympics may complete specific actions to extend their creative prowess. These are tangible, meaningful endeavors in which the Body absorbs, assembles and takes flight, again and again, in relations with others, with things in the environment, assemblage/bringing-together .As physically active humans, we manipulate and explore pathways that are ever-present, being-presented. For example, a figure skater may seek to deliver an Derrida, . PhysicaBody, questioning/able is a prevalent connector and a pathway (evolving) negotiating spaces—creative praxis. At this juncture, praxis (action-reflection) records individuals' innate creative endeavors as they strive toward understanding their environments or those they are propelled to/away. For example, an Olympian may hit a perfect score on home turf at National competitions yet cannot replicate the same routine at the Olympics. Over time, personalized motility (signatures) can be a product of knowledge that seek(s) to connect with hybrid, virtual, local, and unfamiliar spaces. This harmony is a process/reaction/synergy that interacts/connects/creates an equilibrium between the living/lived Body  and thus a place of learning. Within the context of The Body as a Knowledge Incubator™, middle voice is silent, salient, robust and articulative. Middle voice meanders in-between as spoken, performed and/or written literacy. As a Visual Biography it teaches us about ourselves, and others and our place in community. It narrates our storied lives.Physical↔literacy is a site of discovery, proliferated by lived realities. Lived reality—middle In this discussion paper we have tried to unpack the complicated nature of literacy in its physical context. Framing Physical Literacy in its current operational state requires acknowledging its competing narratives within the contexts of Physical Education, Physical Activity, Dance, Sport and Recreation. By unpacking the nature of physical literacy, we discuss the variabilities and entanglement, the in-between spaces. The introduction of The Body as Knowledge Incubator™ purposefully (re)orients the reader to the tangible. Two tangible examples of incubated knowledge include: (1) the body is forever recording interactions that are both meaningful/less to the individual. As the body ages, literacy records the necessity for lifelong learning or understanding of our physical interactions; (2) emancipatory language also provide the body digestible knowledge especially in volatile social and political conditions. Community languages/dialects, student language and dominant languages question conscientization  capabilities. Sadly, in our dualistic world this is not always appreciated. We believe physical literacy is the key literacy on account of our embodied and physical manifestation in the world. Often Physical Literacy is the literacy through which other literacies have to pass. Physical literacy aims to invigorate and elevate the importance of our embodied selves. Through physical activity individuals can not only nurture their own physical literacy but also contribute toward our holistic literacy(ies) that helps us navigate, connect and make sense of ourselves, others and the world around us.difference (Derrida, Our intention within this paper was to provoke thought and highlight the dissonance found at intersections, not to land on one side or the other, but instead to embrace the in-between space, emphasizing this as fertile ground where sense-making and meaning-making can flourish through the embracing of Derrida, , and by Derrida, . We hopeED-M conceptualized the article content. All authors discussed content and wrote and edited the work. 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Evidence, in multiple forms, is a foundation of implementation science. For public health and clinical practice, evidence includes the following: type 1 evidence on etiology and burden; type 2 evidence on effectiveness of interventions; and type 3: evidence on dissemination and implementation (D&I) within context. To support a vision for development and use of evidence in D&I science that is more comprehensive and equitable (particularly for type 3 evidence), this article aims to clarify concepts of evidence, summarize ongoing debates about evidence, and provide a set of recommendations and tools/resources for addressing the “how-to” in filling evidence gaps most critical to advancing implementation science.Because current conceptualizations of evidence have been relatively narrow and insufficiently characterized in our opinion, we identify and discuss challenges and debates about the uses, usefulness, and gaps in evidence for implementation science. A set of questions is proposed to assist in determining when evidence is sufficient for dissemination and implementation. Intersecting gaps include the need to (1) reconsider how the evidence base is determined, (2) improve understanding of contextual effects on implementation, (3) sharpen the focus on health equity in how we approach and build the evidence-base, (4) conduct more policy implementation research and evaluation, and (5) learn from audience and stakeholder perspectives. We offer 15 recommendations to assist in filling these gaps and describe a set of tools for enhancing the evidence most needed in implementation science.To address our recommendations, we see capacity as a necessary ingredient to shift the field’s approach to evidence. Capacity includes the “push” for implementation science where researchers are trained to develop and evaluate evidence which should be useful and feasible for implementers and reflect community or stakeholder priorities. Equally important, there has been inadequate training and too little emphasis on the “pull” for implementation science . We suggest that funders and reviewers of research should adopt and support a more robust definition of evidence. By critically examining the evolving nature of evidence, implementation science can better fulfill its vision of facilitating widespread and equitable adoption, delivery, and sustainment of scientific advances. For every complex problem, there is a solution that is simple, neat, and wrong. — H. L. MenckenContributions to the literatureEvidence in multiple forms is a foundation of implementation science. We describe multiple types of evidence including evidence on etiology and burden, effectiveness of interventions, and implementation within context.We highlight what is missing in current literature on evidence and what is needed to more fully capture and characterize key evidence needed for dissemination and implementation research.For all types of evidence and particularly for evidence regarding dissemination and implementation, complexity and context are essential elements. We provide 15 specific recommendations to advance, specify, and broaden the field’s conceptualization and development of evidence.To fill the evidence gaps, we provide a set of tools and resources that begin to map out the “how-to” for accomplishing research needed to inform more equitable and sustained implementation.Evidence, often informed by a complex cycle of observation, theory, and experiment , is a foDefinitions of evidence and the associated processes (how evidence is used) vary by setting. In clinical settings, evidence-based medicine is “the conscientious, explicit, and judicious use of current best evidence in making decisions about the care of individual patients” . EvidencFor all types of evidence and particularly for type 3 evidence regarding D&I, complexity and context are essential elements –23. BothMuch has been written about the terminology of evidence-based practice and policy. The most widely used term is “evidence-based” practice  evidence on what and for whom in what settings and under what conditions? and (2) When do we have enough evidence for D&I? While the answer to this latter question will always be “it depends,” there are related questions that are useful to consider Table .Table 3DTo facilitate the development and delivery of more equitable and sustainable interventions, we need to expand our thinking about evidence, especially for but not limited to type 3 evidence. We discuss a set of five core interrelated issues about evidence, examining (1) how the evidence base is determined, (2) context, (3) health equity, (4) policy implementation, and (5) audience/stakeholder perspectives. All areas concern some form of research or knowledge gaps in D&I science. The evidence base discussion presents a broader perspective on what is considered evidence; the context, equity, and stakeholder sections cover neglected aspects of implementation science in need of more and higher quality research; and the policy implementation section points to the need for the most pressing gaps in policy-relevant research for D&I. Across these areas, we provide a series of recommendations along with tools and resources for speeding translation of research to practice and policy.Here, we describe ongoing discussions and debates about the uses, usefulness, and gaps in evidence for implementation science, which give way to our recommendations Table . While tThe evidence base for implementation science needs to be broadened to encompass a wider range of study designs, methods, stakeholders, and outcomes. For example, the decontextualized randomized controlled efficacy trial (RCT) that attempts to control for many potential confounding factors is generally considered the gold standard for obtaining evidence on internal validity and contributing to the determination of causality of a given intervention, practice, or treatment . A propeFor clinical and public health practice, the generalizability of an EBI’s effectiveness from one population and setting to another —the core concept of external validity—is an essential ingredient. Systematic review and practice guidelines, which are often the basis for an implementation study, are mainly focused on whether an intervention is effective on average  and have commonly given limited attention to specifying conditions  under which a program is and is not effective –59. For The question of whether an EBI applies involves a set of scientific considerations that may differ from simply knowing average treatment effects. These include balancing of fidelity to the original EBI functions with adaptations needed for replication and scale-up , as wellAdditional evidence is needed on the inter-related concepts of null (ineffective) interventions, de-implementation, and mis-implementation –72. FromEmerging perspectives in data science and causal inference may help advance type 3 evidence. If contextual heterogeneity is the norm, then the scientific task in any one study population is to produce data that address relevance across diverse external settings. Useful methods to do so are becoming available and suggest that the more we know about mediators/mechanisms and modifiers of effects in implementation, the more interpretable findings could be in different settings and populations –76. For The complexity and dynamic nature of implementation necessitate continual attention to context  22, 80. WheThe challenging scientific task of “unpacking” context requires three activities. First, contextual effects in any study setting or across settings and/or systems should be enumerated  should account for historical, cultural, social, and system factors that may shape implementation and the implementation climate, including forms of structural or institutional racism , medical mistrust, institutional and providers’ biases and norms that may create or reinforce biases or inequities, as well as community strengths and assets that may inform implementation efforts. Tools such as critical ethnography can be useful during needs assessment to understand interactions between the ensembles of actors, agencies, interventions, and other contextual variables . When seContext in global settings presents unique considerations, particularly in lower- and middle-income countries (LMICs) and other settings that have limited resources and face numerous structural barriers to health . Among the considerations is the relevant evidence base for implementation—when settings vary tremendously, particularly the social and political context and systems/organizational infrastructure: Do researchers and implementers need to start anew in building the evidence base for implementation, answering many of the questions in Table Transportability theory has been applied to assess whether findings about the effects of an implementation strategy in one setting can be used to infer in another, and if so, whether it is likely to work . Context, Context is also important during the process of broader dissemination of evidence-based approaches. There is a well-documented disconnect between how researchers disseminate their findings (including EBIs) and how practitioners and policy makers learn about the latest evidence . Applyin94Addressing heath disparities and promoting health equity is becoming a more central and explicit focus of implementation science , 97–102.To move towards the goal of achieving health equity, it is critical that implementation science expands the quantity, quality, and types of evidence produced and prioritized, as well as who and what settings are (1) reflected in that evidence (representativeness) and 2) involved in its generation and interpretation (representation). For many health conditions and populations, we have adequate descriptive (type 1) data that can guide what to address . However, we often lack sufficient data on EBIs and strategies that are effective in reducing inequities and/or promoting equity . Often,  involvedA foundational challenge here is that many EBIs were not developed with or tested among settings or populations that experience inequities or with the goal of promoting health equity and may unintentionally contribute to or exacerbate inequities –112. ThiWe need to engage a wider range of partners from lower resource settings earlier and throughout the research process and in meaningful ways to build a broader and more relevant array of equity-focused EBIs that are feasible, acceptable, culturally appropriate, and address root causes. We also need to expand what we “count” as EBIs in public health and clinical research, broadening the focus from a narrower view of individual, interpersonal, and organizational interventions, to also include community, policy, and multi-sector interventions that have the potential to make larger shifts in health inequities. Such broadening of evidence with an eye towards health equity will consider moving beyond a more singular focus on our EBI repositories and including and evaluating existing promising community-defined evidence and interventions , 120. InEquity should be a core criterion for valuing evidence. This value statement should be reflected in priorities of funders, how research questions are framed, how research resources and decision-making are distributed, and how studies are conducted, evaluated, and reviewed. Implementation science has a role in recognizing that a negative consequence of our social and economic systems is the concentration of resources and health. These systems create inequities, so when thinking about closing an implementation gap, we should recognize the context—that such a gap is often an outgrowth of these systems and must be addressed and transformed. Equity needs to be prioritized and made more explicit as part of engagement efforts, which includes consideration of power imbalances (who is and is not involved in making key decisions) and timing of when and how partners are engaged  , 120. ReHealth and social policies, in the form of laws, regulations, organizational practices, and funding priorities, have a substantial impact on the health and well-being of populations and create the conditions under which people can be healthy and thrive- or not , 123. ClPolicy implementation research, which has been conducted since the 1970s across multiple disciplines , 130, sePolicy researchers have utilized a variety of designs, methods, and data sources to investigate the development processes, content, and outcomes of policies. Much more evidence is needed, including which policies work and which do not , how policies should be developed and implemented, unintended consequences of policies, and the best ways to combine quantitative and qualitative methods for evaluation of “upstream” factors that have important implications for health equity . There iSeveral other areas in policy implementation research need attention. First, policy makers often need information on a much shorter time frame than researchers can deliver—this calls for the use of tools such as rapid-cycle research  and rapiThere are multiple audiences of relevance for developing, applying, disseminating, and sustaining the evidence for implementation science . When seKey audience categories include researchers, practitioners, and policy makers Table . ResearcThe relevance and usefulness of evidence vary by stakeholder type Table  151]. R. R151]. Framing is another important factor in considering audiences for D&I. Individuals interpret the same data in different ways depending on the mental model through which they perceive information . For exaThe “how-to” for broadening the evidence base for implementation science will require several actions. First, we need to prioritize the evidence gaps and possible ways of filling these gaps—many ideas are shown in Table Capacity is a core element for building a stronger, more comprehensive, and equitable evidence base. Capacity can be developed in multiple ways, including supporting the “push” for implementation science where researchers are trained to develop the evidence for implementation and skills in evaluation. Evaluation skill building should take into account the principles of realist evaluation, a mixed-methods approach that takes into account multiple contextual variables . There iThere are also macro-level actions that would facilitate a broader and more robust evidence base. For example, funders and guideline developers should adopt a more comprehensive definition of evidence, addressing many of the recommendations outlined in Table Over the past few decades, there has been substantial progress in defining evidence for clinical and public health practice, identifying evidence gaps, and making initial progress in filling certain gaps. Yet to solve the health challenges facing society, we need new and expanded thinking about evidence and commitment to context-based decision-making. This process begins with evidence—a foundation of implementation science. By critically examining and broadening current concepts of evidence, implementation science can better fulfill its vision of providing an explicit response to decades of scientific progress that has not translated into equitable and sustained improvements in population health .


Dalbavancin (DBV) is a long-acting antistaphylococcal lypoglycopeptide that is being increasingly used for long-term treatment of a wide range of subacute and/or chronic infections, mainly osteo-articular infections (OAI). Population pharmacokinetic studies showed that two 1500 mg doses 1 week apart can ensure effective treatment for several weeks. In this scenario, therapeutic drug monitoring (TDM) can be a helpful tool for providing clinicians with real-time feedback on the duration of optimal treatment by measuring drug concentrations over time in each single patient. The aim of this study was to develop and validate a fast and simple analytical method based on the Liquid Chromatography-Isotope Dilution Tandem Mass Spectrometry (ITD LC-MS/MS) technique for measuring DBV concentrations in human plasma microsamples. It will allow an innovative, very convenient and minimally invasive way of sampling. Analysis was performed by simple single-step sample preparation and very short instrumental run time (4 min). Analytical performance met all criteria in terms of specificity, sensitivity, linearity, precision, accuracy, matrix effect, extraction recovery, limit of quantification, dilution integrity and stability under different conditions set by the European Medicines Agency (EMA) for drug quantification by means of bioanalytical methods. The method was successfully applied for measuring DBV concentrations (range = 2.0–77.0 mg/L) in a cohort of patients receiving long-term DBV treatment of subacute and/or chronic infections.  Dalbavancin (DBV)  is a newIn the field of pharmaceutical analysis, High Performance Liquid Chromatography (HPLC) is the most widely used analytical technique for antibiotic assays in biological samples and not only ,14,15,16Currently, few are the methods that have been developed for TDM of DBV. One is based on high-performance liquid chromatography coupled with ultraviolet-visible detection (HPLC-UV-VIS) and has acceptable bias and precision but was tested only in three clinical cases for TDM-based dose optimization of DBV . The othThe aim of this study was to develop and validate a fast and simple analytical method for measuring DBV concentrations in plasma microsamples by means of a liquid chromatography-isotope dilution tandem mass spectrometry (ITD LC-MS/MS) technique using as internal standard the exa-deuterated analog of DBV (DBV-d6), similar to Seraissol et al. . ® Direct system . Drug-free plasma from volunteers was supplied for control purposes by the IRCCS Azienda Ospedaliero Universitaria di Bologna .DBV and DBV-d6  powders were purchased from Sigma-Aldrich  and Clearsynth Labs limited , respectively. The chemical structures of DBV and DBV-d6 are depicted in -Low QC (LQC) = 7 mg/L;-Medium QC (MQC) = 70 mg/L;-High QC (HQC) = 280 mg/L.The DBV stock solution was prepared in MilliQ water at a concentration of 5 mg/mL. The working solution at 500 mg/L was obtained by water dilution of the stock solution and was used as the highest point of the calibration curve. All of the other calibration standards (0.125–5–25–50–250 mg/L) were prepared by subsequent dilution of the working solution with plasma. The final calibration range (0.125–500 mg/L) covered the range of plasma DBV concentrations expected on the basis of the clinical need. Quality control (QC) samples were set up from an independent stock of plasma. The QC samples were set at the following concentrations:IS solution of DBV-d6 was prepared at a concentration of 1 mg/L in methanol. IS and all the other working solutions were frozen at −80 °C.® (Ultra High-Performance Liquid Chromatography) coupled with a quadrupole mass detector 6495c  equipped with an electrospray ion source (ESI). Chromatographic separation was achieved using a ZORBAX Eclipse plus C18 column  . LC-MS/MS analysis was performed by means of an Agilent 1295 UHPLCMobile phase A, 0.1% formic acid in water, and mobile phase B, 0.1% formic acid in methanol were freshly prepared before the analysis. Gradient elution is described in The MS was operated with positive ionization in Multiple Reaction Monitoring (MRM) mode, and the parameters are listed in Chromatographic data acquisition, peak integration and quantification were performed by means of the MassHunter software version 10.0 .Plasma samples (3 μL) were added to 47 μL of ultrapure water to achieve a dilution factor of 17 and then mixed with 150 μL of the IS-methanol solution (1 mg/L DBV-d6). The mixture was vortexed for 15 s and then centrifuged at room temperature at 13,000 rpm for 5 min. Subsequently, 50 µL of the clear supernatant was transferred to an autosampler vial, and a volume of 3 µL was injected into the LC-MS/MS system .The analytical method was validated according to the EMA guidelines for validation of bioanalytical methods . SelectiA selective method should be able to quantify specifically the analyte in a complex mixture without any interference caused by endogenous and/or exogenous components present in the matrix. For testing selectivity, we analyzed 10 different plasma samples coming from different primary sources. The carry-over effect was assessed by checking the eventual presence of peaks in chromatograms of blank plasma samples obtained after running the Upper Limit of Quantification calibrator . Carry-over was considered as suitable whenever peaks were <20% of those recorded for the LLOQ samples.Calibration standards were prepared by spiking the blank matrix over the 0.125 to 500 mg/L range. Linearity of the calibration curve was confirmed by calculating the Percent Relative Error of back-calculated concentration (%RE). According to the fitness-for-purpose approach , to veriPrecision (mean CV%) and accuracy (mean BIAS%) were assessed by extracting and analyzing four concentration levels  for five times both in the same day (intra-day) and in three different days (inter-day).Percent Matrix effect (ME) and Extraction Recovery (ER) were calculated at Low, Medium and High concentration levels by means of the following equations:A = DBV over DBV-d6 mean peak area ratio obtained by injecting water samples (N = 3) spiked at the three concentration levels. after extraction. B = DBV over DBV-d6 mean peak area ratio drug-free matrix extracts (N = 3) spiked at the three concentration levels before extraction.C = DBV over DBV-d6 mean peak area ratio drug-free matrix extracts (N = 3) spiked at the three concentration levels Both ME and ER were tested in 10 different patients’ plasma samples for addressing individual matrix composition variability.sample extracts kept on board at 10 °C for 24 h and frozen at −80 °C for 24 h;plasma samples analyzed after three complete freeze and thaw cycles (from −80 °C to 25 °C).Stability of DBV was assessed by comparing the nominal concentrations of LQC, MQC and HQC with those observed under two different conditions: Stability in the above operating conditions was deemed suitable if DBV concentrations were within ±15% of the nominal value. The presented ITD LC-MS/MS method was tested for measuring DBV concentrations in patients receiving DBV for the treatment of long-term subacute or chronic documented Gram-positive infections . In our study, DBV treatment was started according to the internal protocol consisting of two 1500 mg doses one week apart. Requirement for additional doses was assessed by the treating physician after at least four weeks according to the underlying clinical conditions of each patient. The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the local ethical committee [No. 897/2021/Oss/AOUBo on 29 November 2021]. Informed written consent was waived due to the retrospective and observational nature of the study . The numLC-MS/MS conditions were set for granting good DBV peak shape and quality regardless of the very short chromatographic run time. For this purpose, we selected the ZORBAX Eclipse plus C18 column  , as suggested previously .v/v) and (B) methanol-formic acid  was applied with elution gradient, as described in m/z 909.2 → 340.1 for DBV and at m/z 912.3 → 340.2 for DBV-d6, from the MS/MS fragmentation spectra experimentally obtained (A mobile phase consisting of (A) water-formic acid  was 2.43 min and was very reproducible throughout analytical runs, thus confirming that column reconditioning after the gradient runs was optimal. Consequently, a reconditioning step of 0,5 min in the gradient see  was thouLLOQ sample MRM chromatograms b showed Real sample MRM chromatograms c showed Ten DBV-free plasma samples coming from different sources were scrutinized for checking the eventual presence of interfering peaks in the MRM chromatograms obtained by running the chromatographic gradient. All MRMs of drug-free plasma samples  showed no peak for DBV, thus confirming that carry-over was negligible. This could probably be favored by the step at 95% of mobile phase B see  in the gThe LLOQ was 0.125 μg/mL and corresponded to the lowest point of the calibration curve. Its S/N value was 95.0 and was much higher than the usually considered LOQ of 10.2 = 0.9997. Percent relative error of back-calculated concentrations (%RE) ranged from 18% at LLOQ to an average of 8,7% for all the other calibrators. The calibration curve model using response (= DBV peak area/DBV-d6 peak area) over concentration showed good data fitting (example in Precision (mean CV%) and accuracy (mean BIAS%) results are shown in Percent Matrix effect (%ME) and percent Extraction Recovery yield (%ER) were calculated at Low, Medium and High concentration levels . A signaDBV stability was tested for all of the QC levels in different operating conditions as specified in The DBV concentrations measured by applying the ITD LC-MS/MS method in samples collected from patients receiving DVB for long-term treatment of subacute or chronic documented Gram-positive infections were all within the calibration range. The widespread distribution of DBV plasma concentrations see  below thIn this study, we developed and validated an analytical method for fast, accurate and precise quantitative determination of DBV in human plasma microsamples of 3 μL, by means of an ITD LC-MS/MS technique. Such small microsamples could be obtained by finger or heel pricking and may favor TDM application in long-term treatment regimens. It could be performed during day-hospital accesses or even at home and may minimize the need for venipuncture procedures, the patients’ discomfort and the sample logistic issues ,10,11. 0 and B1 forms of DBV and that the homologous A0, A1 and B2 DBV forms are not taken into account. However, this is not an issue as these forms are not prevalent in the pharmaceutic formulations currently used in humans.Thanks to the high analytical performance, the proposed method met all of the criteria in the EMA guidelines , with thThe method also showed high sensitivity, even potentially much higher than the LLOQ set for validation purposes (at 0.125 mg/L), which is well below the DBV trough levels expected for granting efficacy (namely 4.02 or 8.04 mg/L) . The LLOThe calibration range of our method is extended and dynamic (0.125–500 mg/L) and may cover the entire range of concentrations expected in clinical practice. This makes feasible always measuring DBV concentrations in patients’ samples directly, regardless of them being peak or through values, with no need for sample dilution or re-run even in the presence of very high concentrations.2O (20:80) 27] and/oIn conclusion, we developed a fast, sensitive, accurate, precise and reliable ITD LC-MS/MS method for measuring DBV in human plasma microsamples. The method may be successfully applied for routine TDM of DBV in patients undergoing long-term treatment for subacute and/or chronic staphylococcal infections.