Elucidating underlying mechanisms in subject-specific motor control and perception after amputation could guide development of advanced surgical and neuroprosthetic technologies. In this study, relationships between preserved agonist-antagonist muscle strain within the residual limb and preserved motor control and perception capacity are investigated.Fourteen persons with unilateral transtibial amputations spanning a range of ages, etiologies, and surgical procedures underwent evaluations involving free-space mirrored motions of their lower limbs. Research has shown that varied motor control in biologically intact limbs is executed by the activation of muscle synergies. Here, we assess the naturalness of phantom joint motor control postamputation based on extracted muscle synergies and their activation profiles. Muscle synergy extraction, degree of agonist-antagonist muscle strain, and perception capacity are estimated from electromyography, ultrasonography, and goniometry, respectively.P < 0.005–0.05) between sensorimotor responses and residual limb agonist-antagonist muscle strain. Identified trends indicate that preserving even 20–26% of agonist-antagonist muscle strain within the residuum compared to a biologically intact limb is effective in preserving natural motor control postamputation, though preserving limb perception capacity requires more (61%) agonist-antagonist muscle strain preservation.Here, we show significant positive correlations  and motor control and perception in lower limb amputees, with some receiving a myoneural interface intervention. The authors report that the degree of AMS within the residual limb is associated with preserved motor control and perception. Among these sensory organs, proprioception is primarily mediated by mechanoreceptors called muscle spindles and Golgi tendon organs which sense muscle length, speed, and tension3. Proprioceptive neural signaling relies on both microscale mechanotransduction processes4, and macroscale biomechanically-functional tissue architectures1. The realization of such an architecture in a person with biologically intact limbs is implemented by mechanically-coupled antagonistic muscles spanning an articular joint that enables afferent signaling from the mechanoreceptors corresponding to limb movements through agonist-antagonist muscle strain (AMS).Proprioception is possible due to the presence of sensory organs within peripheral tissues including muscles, tendons, joint capsules, and skin6. However, in the absence of visual feedback, postural responses and balance during walking remain challenging for persons with leg amputation8, which may indicate that motor control and proprioceptive percepts are significantly altered in persons that have undergone a conventional amputation procedure11. Toward better, more biomimetic control of an external prosthesis, invasive nerve interfacing using artificial electrical stimulation has shown great potential in restoring cutaneous14 and proprioceptive sensation12. However, due to the complexity of afferent signaling through artificial nerve stimulation, and the relatively limited resolution of state-of-the-art implantable devices, it can be challenging to engineer stable neuroprosthetic interfaces that offer natural cutaneous and proprioceptive percepts.The conventional standard-of-care amputation paradigm permanently disrupts the anatomical and neuromechanical principles of AMS, resulting in perturbed proprioception in people living with limb loss. Many ongoing efforts to restore locomotion for persons with leg amputation involve motor intent classification strategies based on electromyography (EMG) and intrinsic prosthetic signals15. Conventional myocutaneous flap amputation procedures prioritize creating enough muscle padding for prosthetic socket fitting16. To enhance neuroprosthetic control, amputation paradigms seeking further reconfiguration of residual limb soft tissues have been developed, including Targeted Muscle Reinnervation (TMR)20, Regenerative Peripheral Nerve Interfaces (rPNIs)23, and the Agonist-antagonist Myoneural Interface (AMI)27. Each of these techniques have been demonstrated in combination with neuroprostheses24 for the enhancement of prosthetic control. Nevertheless, neuroprosthetic performance is a system-level evaluation that depends on multiple factors such as subject-specific inherent capacities of residual limb motor control and phantom limb perception, engineered functional feedback, presence of visual feedback, and choice of the neuroprosthetic control paradigm. Consequently, there is a clear and present need to uncover the fundamental nature of how surgical residual limb reconstruction alone impacts clinical outcomes after amputation.As an alternative approach to neuroprosthetic interface design, surgical methodologies to reconfigure residual limb soft tissues may lower the burden of engineering and offer a more efficacious, biomimetic motor control strategy while also providing feedback from the amputated limb via biological sensory organs22. The study focuses on the impact of AMS preservation within the residual limb on motor control and phantom limb perception. We hypothesize that enhanced levels of residual limb AMS will improve motor control naturalness and proprioceptive perception postamputation in persons with transtibial amputation. To evaluate this hypothesis, the study clinically evaluates the naturalness of motor control and limb perception capacity during ankle and subtalar joint movements without visual or any other functional feedback. We collected muscle electromyography patterns and two degrees-of-freedom (2-DoF) kinetic data during bilateral, mirrored movements between the intact and phantom ankle-foot limbs of each subject. For these mirrored movements, we assessed the degree of residual limb AMS using ultrasonography. Because motor control in biologically-intact limbs is executed by the activation of combinations of muscle synergies, we evaluate motor control naturalness and limb perception of amputees using muscle synergy analysis30. The study findings support the hypothesis that enhancing AMS in the residual limb improves motor control naturalness and perception after amputation, underscoring the importance of surgical techniques such as the AMI that create a residuum tissue structure that preserves agonist-antagonist muscle dynamics.In this study, we investigate motor control and phantom limb perception capacities of 14 clinical research subjects having unilateral transtibial amputation spanning a range of ages and etiologies. Of the 14 participants, 7 subjects had received the AMI amputation, and 7 had undergone a non-AMI amputation. The transtibial AMI amputation comprises the surgical creation of dynamic agonist-antagonist muscle pairs for the enhancement of AMS. One muscle pair is constructed for the ankle joint comprising the lateral gastrocnemius linked to the tibialis anterior, and a second muscle pair for the subtalar joint comprising the peroneus longus linked to the tibialis posteriorhttps://clinicaltrials.gov/). The relationship of the present work to that trial is to obtain preliminary data and an algorithmic framework—a muscle synergy model—and thus inform our assessment of the pre-specified outcome measures for NCT03913273. Computational tools such as the model investigated in the present work are a critical part of studying and quantifying voluntary motor control postamputation in free space, ambulatory ascent and descent of stepping stairs while wearing a prototype multi-degree-of-freedom prosthesis, and potential for closed-loop prosthetic control by functional electrical stimulation.The present work begins to investigate one outcome measure—economy of motion—of our ongoing clinical trial, NCT03913273, although we do not report any pre-specified endpoints of that trial in the present work. In overall scope the NCT03913273 trial investigates if AMIs can (i) improve voluntary free-space prosthetic control, (ii) improve voluntary and involuntary (reflexive) prosthetic terrain adaptations, and (iii) serve as a bidirectional human-device interface after transtibial amputation (31). Exclusion criteria included one more of the following underlying health conditions: cardiopulmonary instability manifest as coronary artery disease, chronic obstructive pulmonary disease, extensive microvascular compromise, as well as persons who are pregnant and/or active smokers.All data in the present study, were collected at the Massachusetts Institute of Technology (MIT) under IRB approval from our Committee on the Use of Humans as Experimental Subjects (protocol 1812634918). All participants signed informed consent forms prior to data collection. The work followed the same prospective, non-randomized study design described in NCT03913273. The time period of recruitment and data collection was June 12, 2019 through September 19, 2021. Eligibility criteria included transtibial amputee subjects within an age range of 18 to 65 years, a fully healed amputation site, proficiency in the use of a standard lower-extremity prosthesis, and capability for ambulation with variable cadence (K level 3 and 425 (7/14), conventional16 (6/14), and Ertl osteomyoplasty33 (1/14). AMI amputation surgeries had been done according to Partner’s Institutional Review Board protocol p2014001379 as in previous reports25.Table 23. For the ankle joint AMI construct, the tibialis anterior (TA) was linked to the lateral gastrocnemius (GA), and for the subtalar joint AMI construct, the tibialis posterior (TP) was linked to the peroneus longus (PL). The AMI amputation aims to emulate physiological antagonistic actuation between the residual limb muscles to restore AMS  and unaffected biologically intact limbs . A 2-DoF goniometer was also placed on the posterior aspect of the unaffected BIO limb spanning the ankle-foot complex 36. The NMF was continued until the change in VAF in 50 consecutive iterations was less than a tolerance of 36. To enable intrasubject comparisons of motor commands, the average vectors of synergy activation profiles The motor control of residual limbs was evaluated by muscle synergy extraction from muscle activation patterns during discrete ankle and subtalar joint movement trials. Subjects were asked to sequentially make PF, IN, DF, and EV movements of both the residual (phantom) and biologically intact limbs. The order of discrete movement trials was not randomized in an effort to identify existing motor capabilities for the 4 principal movements as accurately as possible. Discrete movements were repeated 40 times. We used a generally accepted mathematical model0 vectors were added to muscle synergy and synergy activation vectors to match the dimensionality of vectors.We quantified the naturalness of muscle synergy and synergy activation profiles of AMI and CTL groups by computing their similarities to the average normalized values of the BIO group. Specifically, a muscle synergy of one subject was considered to correspond to a muscle synergy of another subject when the maximum of the scalar products was found among others. After sorting the muscle synergy vectors, the representative muscle synergy vectors of the BIO group were determined as the normalized average muscle synergy vectors of the BIO group. Finally, the naturalness of one’s muscle synergy was calculated by plotting the mean scalar products with the representative of the BIO group. Similarly, the naturalness of one’s synergy activation vectors was calculated by plotting the mean scalar products with the normalized average synergy activation vectors of the BIO group. For the BIO group, a leave-one-out procedure was used for computing their dot products. The universal number of synergy vectors for similarities analysis was unified to 3 muscle synergy vectors, which was the number of synergy vectors of all subjects in the BIO group. When fewer synergy vectors were identified previously by synergy extraction procedures, ϕij is the margin in synergy activations to have distinguishable patterns between targeted i and j discrete movements; Ttask,i is the interval time of i discrete movement. Note that the first term in the right-hand side of the equation is the angle between two synergy activation average vectors (tolerance); the second and third terms are variability in motor commands corresponding to the two discrete movements. The thresholds of 0 were selected for synergy activation margin to determine robustly decoupled discrete movements.We quantified the robustness of ankle and subtalar volitional control based on the degree of decoupling between synergy activations for different target movements. The angle between two average vectors of synergy activations for two different target movements indicates the tolerance to variance in motor commands of two corresponding targeted movements. When the tolerance of motor commands is larger than the expected variance, it indicates that a subject can reliably produce distinguishable synergy activation for two discrete movements of interest. Therefore, the margin of synergy activation is given as37. The same initialization and iteration protocols were utilized as those of muscle synergy extraction procedures for the rest of decomposition procedures. Given the revised NMF, the arbitrary muscle activation patterns can be reflected into the muscle synergy space of the discrete ankle and subtalar joint movements. This reflected time-varying coefficients at time t, 0 vectors were added in We decoded the motor intents from arbitrary muscle activation patterns based on the extracted muscle synergy and synergy activation average vector of the 4 principal movements . The time-varying coefficients of the arbitrary muscle activation patterns were decomposed by revised NMF, fixing synergy vectors as the extracted muscle synergy from discrete movement trials during NMF iterationsj-th quadrant of ith trajectory in jth quadrant, respectively; We quantified simultaneous multi-DoF motor controllability by investigating the transitions in directionality of motor intent during 10 cycles of the drawing-a-circle tasks. First, the 40. Speed-accuracy tasks comprised 10 repetitions in each of discrete PF, DF, IN, EV movements in a random order for each of 5 time-interval settings. The motor control performance was analyzed based on the tracking errors between the decoded motor intent j discrete movement Spatiotemporal motor control was evaluated from two metrics, motor control performance and economy of motion under increasing time constraints from 2.0 s to 1.5 s, 1 s, 0.8 s, and 0.5 s. For the visualization, an index of difficulty (ID) of speed-accuracy task for each time constraint (2.0–0.5 s) was calculated as the logarithm of the inverse of the time constraint and scaled to range from 0 to 1  for DF-PF and IN-EV. The mirrored perceived phantom limb positions were measured by goniometry from the subjects’ BIO limb and were normalized by the ROM of the BIO limb , paired one-tailed t-tests were used at a significance level of α = 0.05, as all motor control data did not violate the data normality. To consider between-subject residual limb differences (AMI:CTL), unpaired two-tailed t-tests were used at a significance level of α = 0.05. Interactive effects between limb subgroups (AMI:CTL x affected:unaffected limb) were analyzed by 2-way ANOVA at a significance level of α = 0.05. The full statistics are reported in the Supplementary Table No statistical methods were used to predetermine sample size a priori, but effect sizes were determined for the main outcomes using Cohen’s Further information on research design is available in the 30. We evaluated the naturalness of motor control of amputees by computing similarities between muscle synergies and their activation profiles for residual limbs and biologically intact limbs. We used a linear synergy model to extract muscle synergy and activation profiles from recorded EMG using a dimensionality-reduction technique30. The subjects performed discrete ankle and subtalar motion tasks that were assigned in the chronological order of PF-IN-DF-EV, which was not randomized in an effort to identify existing muscle synergies for the 4 principal movements as accurately as possible for the forthcoming analyses. The extracted muscle synergies and average vectors of synergy activation profiles for all 28 limbs are shown in Fig. , showing altered GA and TP coordination profiles . Relatively low values of the critical degree of AMS . The results provide evidence in support of the degree of AMS within residual muscles enabling natural, discrete motor control.The muscle synergy similarity (m.s.s.) and synergy activation similarity (s.a.s.) between each subject’s residual limb and the average profiles across all 14 subjects’ biologically intact limbs was plotted against the degree of AMS Fig. , e. Botht = 3.30, P < 0.01, s.a.s.: t = 2.99, P < 0.02), significant differences for CTL:BIO-C comparisons , and no significant differences for AMI:BIO-A comparisons . Further, subgroup analyses amongst the 28 limbs revealed significant interactive effects (AMI:CTL × affected:unaffected limb) for muscle synergy similarity and synergy activation similarity . Together, the results suggest that advanced amputation procedures that actively preserve even a small degree of AMS may effectively preserve natural discrete motor control after amputation.Further analyses Fig.  found siHigh reliability of motor control that is consistent and cohesive, even in the absence of visual or other functional feedback, is anticipated to provide stable neural signaling and thereby enable neuroprosthetic control. To assess robust motor control for ankle and subtalar joint movements, we investigated the variability in synergy activation profiles for each movement Fig. . Synergy37. We then computed motor intents from the synergy activations based on the average vectors of synergy activation profiles of the 4 principal ankle and subtalar joint movements, transforming into the To evaluate simultaneous multi-DoF motor controllability, we asked the subjects to attempt ‘drawing a circle’ with mirroring, of their phantom foot and biologically intact foot by simultaneously controlling their ankle and subtalar joints. We decoded the measured muscle activations into synergy activations , and a relatively low R2 = 0.89). Significant interactive effects  were found for 2-DoF motor controllability between amputation subgroup and limb category (AMI:CTL × affected:unaffected limb). Also, significant differences in 2-DoF motor controllability were found between CTL and BIO-C limbs  and between AMI and CTL limbs , whereas no significant difference was found between BIO-A and AMI limbs .The 2-DoF motor controllability plotted against the degree of AMS Fig.  showed sTogether, our results suggest that AMS within residual muscles enhances the decoupling and stabilization of the motor behaviors for discrete ankle and subtalar joint movements, allowing robust, simultaneous 2-DoF motor control of the residual limbs without visual or other functional feedback. Further, our results imply that amputation procedures that actively preserve biological AMS may effectively preserve multi-DoF motor control after amputation.40. We imposed time constraints, of 2.0, 1.5, 1.0, 0.8, or 0.5 s, within which subjects were asked to perform the 4 discrete ankle and subtalar movements  across all IDs .As visualized by 4 different colors in Fig. IDs Fig. , d. We fIDs Fig. , f showeOur results suggest that a higher degree of preserved AMS can provide a person with an amputation more sustained economy of motion, with minimized wasted movements, during spatiotemporal motor tasks. Conversely, a subject with limited AMS was less able to maintain efficacious motion and ‘wandered’ more during spatiotemporal motor control performance tasks shown as a low economy of motion. We speculate that the afferent feedback inherent in residual limb AMS may reduce ‘trembling’ of neuroprosthetic control in the absence of visual or other functional feedback to enable improved neuroprosthetic control, as will be further discussed in the Discussion section.1 exert a strong influence on both motor control41 and proprioceptive limb perception42. If the degree of residual AMS is a critical neurophysiological determinant underlying natural motor control preservation after limb amputation, we postulate that AMS should also contribute to preserving proprioceptive phantom limb perception. To explore this experimentally, we asked the subjects to move and vary their phantom foot positions to 25, 50, 75, and 100% of their DF or PF range of motion, and to 50 and 100% of their IN or EV range of motion, while mirroring phantom limb perception with their biologically intact foot. We assessed the intended phantom limb positions  - are shown in Fig. 33, exhibited high limb perception capacity (0.60) and high capability to vary the position of his phantom foot. Consistently, when compared to the muscle dynamics presented by AMI-1, CTL-3, and CTL-5, the ultrasound examination of CTL-5 revealed 2-DoF ‘joystick-like’ coupling between antagonistic muscle pairs that were distributed across the inferior aspect of his residual limb, as illustrated in the comparison shown in Fig. We estimated the values of limb perception capacity based on the psychometric function defined by plotting dom Fig. . Data foP < 0.005) across all 14 residual limbs. However, the R2 = 0.45) compared with the values of Limb perception capacity plotted against the degree of AMS Fig.  showed aP = 0.91, CTL: P = 0.42), synergy activation similarity , 2-DoF motor controllability , or limb perception capacity  and time since amputation for the 14 residual limbs. Also, no significant differences between the AMI and CTL subjects were seen in the maximum EMG values recorded from the four target muscles , average scar tissue cross-sectional area , quantified with ultrasonography43, or average phantom limb pain score .We have gathered and analyzed clinical metrics including correlations between time since amputation and sensory-motor responses, maximum EMG values, average cross section of scar tissue, and average phantom limb score  level48. To the best of our knowledge, the present work is the first to address the relationships between residual muscle biomechanics and muscle synergies in persons with major limb amputation. Together, our results suggest that AMS can provide a characteristic, readily ascertainable residual limb structural feature that can help to explain the variability in amputation outcomes and contribute together with other non-AMS factors including neuroplasticity, proprioceptive memory, neural signaling deficits, subject-specific perceptive responses, differences in scar tissue formation postamputation, and other biomechanical factors within the limb.Muscle synergies in non-amputated humans reflect reflexive and afferent neural feedback in a manner dictated by musculotendon length changes and joint biomechanics49. When musculature distal to the amputation level is intact and viable, it may be harvested on a neurovascular leash during the amputation procedure, and AMS preservation can then be surgically implemented through muscle pair coupling and mechanical fixation of the construct to the surrounding fascia and muscle within the residuum50. If musculature distal to the amputation level is not viable, then AMS preservation may be implemented by constructing a native AMI using large vascularized muscle with TMR nerve reinnervation15, or alternatively by constructing a regenerative AMI from reinnervated muscle grafts51.In this study, we address the beneficial outcomes of surgical paradigms that can actively preserve AMS during transtibial amputation. AMS preservation may also improve motor control and proprioceptive percepts for patients who undergo amputations at other anatomical levels. The AMI amputation procedure has now been conducted on over 30 patients at the transtibial, transfemoral, transradial and transhumeral levels to enable improved motor control and sensory perception in a broader population of amputee subjects15. Another consideration is operative time; AMS preservation during amputation requires a somewhat extended operation time52, and as such may not be appropriate in some scenarios. In other cases, such as life-threatening emergencies, AMS may be excluded initially and introduced during a revision surgery, which is often performed to treat phantom limb pain or neuroma postamputation20.In some instances, it may not be feasible to surgically implement all agonist-antagonist muscle couplings to fully emulate intact-limb dynamics due to limited physical volume of the residuum skin envelope . In this case, the AMI procedure may be applied to only major agonist-antagonist muscle pairs while other surgical strategies such as rPNI and TMR are applied to the remaining musculature sites and transected nerves. Thus, through an integration of AMI, rPNI, and TMR techniques, the amputation procedure can be designed to improve motor control and sensory perception for patient-specific functional limb restoration53 for in-socket EMG recordings from residual muscles. We developed and investigated film-thick electrodes54 and a neuroprosthesis55, with which we are exploring AMI amputee subjects’ capabilities for level ground walking and terrain adaptation. We are also integrating AMI constructs with an osseointegration implant, wherein a bone-anchored mechanical conduit can allow percutaneous passage of 16 implanted leads for electrical stimulation of, and recording from, an amputee subject’s residuum muscles56. In this approach implanted EMG electrodes are placed onto each AMI muscle with each electrode’s wire leads passing through the osseointegrated implant.In this paper, we demonstrate the benefits of preserving AMS on motor control and sensory perception in free space using a synergy analysis that did not require a physical external prosthesis or a specific control system. In the EMG control of external powered prostheses, a critical challenge is the surface EMG electrodes needed for neuroprosthetic applications. Various research groups and companies are developing flexible, thin electrodesAMSc, derived through a combination of mathematical, biomechanical, and clinical data. Consequently, our findings offer new, meaningful insights into motor and sensory perturbations by people living with major limb amputation. With further refinement, the concept of a critical degree of AMS may elucidate fundamental factors underlying clinical outcomes after amputation and inform future amputation paradigms and neuroprosthetic system designs.In this study, we present a platform that combines muscle synergy analysis with biophysical and biomedical sciences to clinically demonstrate the impact of AMS on physiological motor control after limb amputation in 14 amputee subjects. Naturalness of motor control is investigated using the extracted muscle synergies and their activation profiles. Our approach reveals that preserving a relatively low degree of AMS in the residuum  is effective in preserving natural motor control postamputation, though preserving limb perception capacity requires a higher degree (61%) of AMS preservation. Our findings suggest that AMS-amputation strategies are one way to enable more effective and biomimetic sensorimotor control postamputation. Previously, the reasons underlying motor control and proprioceptive perception postamputation were poorly understood. The unique circumstances of our study population and dataset, which comprises 28 AMI and CTL limbs - their post-amputation residual limbs and biologically intact limbs—allowed the discovery of a fundamental index. The present approach provides a predictive index of postamputation outcome, For the motor tasks performed, our study provided evidence to support the hypothesis that the degree of AMS within the transtibial residuum is the neuromechanical determinant of subject-specific motor control and proprioceptive preservation. The main limitation of our study was its small population size—14 transtibial amputees including 7 AMI and 7 non-AMI participants. We anticipate that future, larger studies will further elucidate the correlations established herein and provide further insight into how a multiplicity of factors such as age, time since amputation, body habitus, and amputation etiology may impact sensorimotor responses.49. To this end, basic research advances are necessary in physiological actuators such as biocompatible synthetic actuators57, intramuscular sensors60, and stimulators that can be implanted in series with residual muscle end organs.Emulating the natural biomechanics of muscle interaction to impart a high degree of AMS in residual limbs requires a sophisticated surgical design. Surgical considerations include physiological muscle tensions in mechanical couplings, balanced muscle force capacities, and minimizing the mechanical impedance to enable freely moving agonist-antagonist muscle dynamics. In practice, taking these multiple factors into account during the amputation procedure is challenging. In this study, the large variance in AMS across the AMI cohort underscores the need for further optimization of AMI amputation technique to enable a more consistent AMS clinical outcome. More broadly, an exciting area of future research would be the development of residual limb architectures that enable direct computer control of mechanoneural transduction4. However, the motor control consequences of altered afferent signals by the macroscale reconfiguration of biomechanically-functional tissue architectures is not yet understood. Here we document the effects of modified afferent signaling from distinct functional tissue architectures of the transtibial residuum by revealing the neuromechanical determinant, and the sensitivity of its impact on the CNS, to subject-specific residual motor control and phantom limb perception. Toward the design of biomimetic neural interfaces, we wish to underscore the value of surgical techniques that create a residuum tissue structure that preserves natural neuromusculature and biomechanical function.The importance of mechanoneural transduction within sensory organs for afferent signaling is well knownDescription of Additional Supplementary FilesSupplementary InformationSupplementary Data 1Supplementary Data 2Supplementary Data 3Peer Review FileReporting Summary


Trans-phalangeal and trans-metatarsal amputation, collectively termed ‘minor amputations’ are important procedures for managing infections of diabetes-related foot ulcers (DFU). Following minor amputation, international guidelines recommend a prolonged course of antibiotics if residual infected bone on intra-operative bone samples are identified, but the quality of the evidence underpinning these guidelines is low. In this study, we examined the concordance of microbiological results from proximal bone cultures compared to results from superficial wound swabs in relation to patient outcomes; with the aim of determining the utility of routinely obtaining marginal bone specimens.Data was retrospectively collected on 144 individuals who underwent minor amputations for infected DFU at a large Australian tertiary hospital. Concordance was identified for patients with both superficial wound swabs and intra-operative bone samples available. Patient outcomes were monitored up to 6 months post-amputation. The primary outcome was complete healing at 6 months; and secondary outcome measures included further surgery and death. Mann Whitney U testing was performed for bivariate analyses of continuous variables, Chi-Squared testing used for categorical variables and a logistic regression was performed with healing as the dependent variable.P = 0.89). Revascularisation during admission  and amputation of the 5th ray  were independent risk factors for non-healing.A moderate-high degree of concordance was observed between microbiological samples, with 38/111 (35%) of patients having discordant wound swab and bone sample microbiology. Discordant results were not associated with adverse outcomes (67.2% with concordant results achieved complete healing compared with 68.6% patients with discordant results; There was a moderate-high degree of concordance between superficial wound swab results and intra-operative bone sample microbiology in this patient cohort. Discordance was not associated with adverse outcomes. These results suggest there is little clinical utility in routinely collecting proximal bone as an adjunct to routine wound swabs for culture during minor amputation for an infected DFU. Globally, one person loses a limb every 30 seconds, usually as a direct consequence of an infected diabetes-related foot ulcer (DFU) . Trans-pThe diagnosis and management of DFU infections and osteomyelitis is dependent on clinical signs and symptoms, in conjunction with blood laboratory, microbiological, and radiological evidence of infection. However, ascertaining and interpreting microbiological sample results is challenging . SourcesIndeed, international guidelines recommend 2–5 days of antibiotic therapy following amputation with no residual infection. By contrast, for residual infected bone, a period of intravenous therapy is recommended, followed by oral antibiotics to complete 4–6 weeks treatment .The utility of obtaining a marginal bone specimen in determining which patients need further surgical or antibiotic management was identified as a knowledge gap in recent international guideline documents . While sIn this single centre retrospective study, we aimed to identify the degree of concordance between microbiological results of proximal bone culture collected routinely following minor amputation for infected DFU, in relation to results from superficial ulcer swabs and correlating with clinical outcomes in terms of complete healing, subsequent further operations, and major limb amputation. The purpose of this was to address the utility of routine acquisition of marginal bone specimens in determining which patients require further surgical or antimicrobial management. We hypothesised that the routine collection of marginal bone for culture has little clinical utility.Fiona Stanley Hospital (FSH) is a 783-bed tertiary hospital with a specialised multidisciplinary diabetes foot unit (MDFU). This multidisciplinary team comprises of endocrinologists, vascular surgeons, infectious diseases (ID) physicians, podiatrists, and community liaison nurses. The team manages complex diabetes-related foot complications across both inpatient and outpatient settings. A medical photographer attends all outpatient clinics to document ulcer site, size, and the presence of a healed wound. Outpatient wound care and, if required, parenteral antibiotics are provided by a single ambulatory nursing service.The current study is a sub-study of the Audit of Multidisciplinary Diabetes Foot Unit services at Fiona Stanley and Fremantle Hospitals. Ethical approval was obtained from South Metropolitan Health Human Research Ethics Committee (RGS0000003204). To compare outcomes for patients hospitalised for diabetes related foot disease over time, two time periods of one year duration were studied in 2015 and 2019. For both the 2015 and 2019 cohorts this included hospitalisations during 52-week periods from the first week of February in each year. Between 2015 and 2019, clinical practice remained consistent, as endocrinology, ID and vascular surgery clinical leads remained the same.The inclusion criteria for this study were as follows: 1] a diagnosis of diabetes, 2] admission for an infected diabetic foot ulcer and 3] minor amputation performed during admission. A minor amputation was defined as either a trans-phalangeal or trans-metatarsal amputation of single or multiple digits. Patients undergoing more complex surgeries including proximal forefoot amputations, revision of previous amputation sites or major lower extremity amputations were excluded.In addition to the electronic medical record (EMR), data were also collected from laboratory and radiography databases. Demographics including age, sex, type of diabetes, glycaemic control, smoking history, diagnosis of chronic kidney disease and presence of Charcot neuroarthropathy were recorded. Glycaemic control was assessed with a pre-operative glycated haemoglobin (HbA1C). Smoking history was documented as never smoker or a history of previous/current smoking. Presence of chronic kidney disease (CKD) was judged based on the pre-operative eGFR (estimated glomerular filtration rate), and renal replacement therapy was noted. The history of an amputation was captured in the EMR and confirmed with review of operative notes. The requirement for an angioplasty during admission was also recorded.Bone specimens were collected intraoperatively. Following bone transection, proximal bone samples were collected using a sterile ‘bone-nibbler’ and sent for culture. Our local standard of practice was for 2-weeks of oral antibiotics following the amputation, unless there was clinical evidence of residual soft tissue infection or involvement of adjacent osteoarticular structures. To define infection severity prior to the amputation, we applied the Infectious Diseases Society of America/International Working Group on the Diabetic Foot infection grading system (IDSA/IWGDF) , 10. AmpWe defined microbiology results as concordant if a swab and bone specimen were sent to the laboratory and the same organism(s) were isolated. We also considered concordant results if the bone cultures were negative, but an organism was isolated from superficial swabs. Pre-operative and post-operative antibiotic choice and planned duration was recorded. If discordance between swab and bone specimen was noted, the change in antibiotic choice was recorded.Patient outcomes were assessed from the EMR up until six months post initial amputation. The primary outcome measure was the presence of complete healing by the end of the follow-up period. Secondary outcome measures included death, progression to a major amputation and the need for further surgery. If within this six-month period, a patient required intervention for a new ulceration within the immediate vicinity of the original amputation site, this was deemed contiguous with the original infection. Complete healing was defined as complete closure of the primary wound with no evidence of ulcer relapse within six months of the initial surgery. Further surgery was defined as further surgical intervention to the original amputation site, or a site within the immediate vicinity of the original infection.P-value < 0.1 were eligible for inclusion and retained in the model if P < 0.05. The most parsimonious model was chosen on the basis of the Akaike Information Criterion. All statistical analyses were performed using R  0.37 [0.13–0.96], P = 0.04) and amputation of the 5th ray  were independent risk factors for not healing.Bivariate analyses of factors associated with healing are shown Table . There wCurrent guidelines do not encourage routine collection of superficial wound swabs in the management of infected DFUs , 6. CertFurther to this, international guidelines currently recommend where possible, the use of intraoperative bone culture or histopathological sampling (when available) to accurately diagnose the presence of an infected DFU and osteomyelitis , 6. The Where discordant results were obtained, intra-operative bone samples were more likely to be polymicrobial with higher proportions of CoNS, ESCAPPM, coliforms and enterococci. These findings are likely to be due to differences in how the laboratory handles swabs and tissue specimens. All organisms identified from bone specimens are formally identified to the species level, and susceptibility profiles reported. Notwithstanding these handling differences, these findings are likely related to the risk of intra-operative sample contamination during collection .Unsurprisingly, these results demonstrated that the need for an inpatient angiogram had a higher association with non-healing. Ischaemia related to peripheral arterial disease is a well-known risk factor for non-healing ulcers, infection, and further amputation . If a paThis study has limitations. The sample size was relatively small, and due to its retrospective nature, relied on the contents of the EMR. As such, outcomes of some patients had to be excluded due to loss to follow-up or absence of documentation in the EMR. Histopathology was not performed on any patient, which limits any conclusion on the utility for this modality in defining patients at risk for poor outcomes. While histopathological assessment of bone samples may be useful in correlating microbiology from swab and bone samples to further ascertain the utility of intra-operative bone sampling, its practicality and reliability needs to assessed in a future study.This study demonstrates a moderate-high degree of concordance between superficial wound swab and intra-operative bone sample microbiology. Discordance between the microbiological samples did not result in significantly worse outcomes and this was independent of changes made to the antibiotic choice following identification of discordance suggesting little clinical utility in routinely collecting proximal bone for culture during minor amputation.As a result, choice and duration of antibiotic therapy may be determined by superficial wound swab cultures and clinical correlation with evidence of residual soft tissue infection or osteoarticular involvement of adjacent structures. Further research should evaluate the clinical utility, reliability, and cost-effectiveness of such an approach in combination with histopathological assessment.


Skeletal muscle metastasis from lung cancer is a rare phenomenon. In this case report, FDG PET/CT imaging detected unexpected metastatic spread in skeletal muscles from lung cancer. In lung cancer, the multiple locations frequent metastases are represented by the adrenal glands, the liver, the skeleton and the brain. In this sense, PET certainly has an advantage compared to other imaging methods because the oncological tomoscintigraphic study is always performed with a total body scan  and therefore also allows an evaluation of all body districts, with the exception of the brain, to search for remote locations of the disease (M staging). Several studies have demonstrated the ability of PET to identify distant metastases with high specificity, superior to what can be achieved with conventional imaging, including CT  (Schrevens and Lorent In this case report, FDG PET/CT imaging detected unexpected metastatic spread in skeletal muscles from lung cancer.A 51-year-old man with multiple subcutaneous nodular lesions of suspected oncological origin referred to our Nuclear Medicine Department in order to find out the culprit primary lesion.The 18F-FDG PET/CT study demonstrated an increased FDG uptake in the primary tumor located in the upper lobe of the left lung, bilateral adrenal glands metastasis, multiple osteolytic bone lesions and multiple hypermetabolic nodular lesions in various skeletal muscle Figs. , 3, alsoSurgical biopsy from hypermetabolic muscle mass in the posteroinferior thoracic wall revealed to be a metastatic site from adenocarcinoma of lung origin staining with cytokeratin-7, but thyroid transcription factor-1 and cytokeratin-20 were negative. Also thyroid transcription factor-1 was negative, and this factor may occur in 28% of cases with lung adenocarcinoma (Yatabe et al. FDG PET/CT scan in this case demonstrated to be an excellent imaging method in detecting muscle involvements in lung cancer patients, which are a rare site of metastatic involvement from adenocarcinoma lung cancer (Giorgio et al.


Goal: To achieve high-quality comprehensive feature extraction from physiological signals that enables precise physiological parameter estimation despite evolving waveform morphologies. Methods: We propose Boosted-SpringDTW, a probabilistic framework that leverages dynamic time warping (DTW) and minimal domain-specific heuristics to simultaneously segment physiological signals and identify fiducial points that represent cardiac events. An automated dynamic template adapts to evolving waveform morphologies. We validate Boosted-SpringDTW performance with a benchmark PPG dataset whose morphologies include subject- and respiratory-induced variation. Results: Boosted-SpringDTW achieves precision, recall, and F1-scores over 0.96 for identifying fiducial points and mean absolute error values less than 11.41 milliseconds when estimating IBI. Conclusion: Boosted-SpringDTW improves F1-Scores compared to two baseline feature extraction algorithms by 35% on average for fiducial point identification and mean percent difference by 16% on average for IBI estimation. Significance: Precise hemodynamic parameter estimation with wearable devices enables continuous health monitoring throughout a patients’ daily life. I.Hemodynamic parameter estimation with wearable devices enables continuous health monitoring in outpatient settings, thus granting diagnostic insight throughout varying contexts of a patient's daily activities. Not commonly captured in clinical settings, this information contributes to the early detection of life-threatening illnesses, advanced fitness tracking, and emotional regulation All physiological waveforms that capture blood flow or represent heart beats – including photoplethysmography (PPG) Dynamic time warping (DTW) shows potential for simultaneously identifying all target fiducial points. This established technique compares the likeness between two signals to produce sample-to-sample mappings between them without any prior knowledge on the their underlying physics In this work, we propose Boosted-SpringDTW as an automated approach towards comprehensive feature extraction of physiological waveforms. Particularly, our proposed framework overcomes the aforementioned challenges associated with the two common sub-tasks required for analyzing a stream of quasi-periodic physiological signals: 1) segmentation into cardiac cycles and 2) identification of all fiducial points. Given a single template of a typical cardiac cycle, our framework conducts a probabilistic decision-making process to precisely identify the true start and endpoints within a waveform stream by leveraging both minimum domain-specific characteristics of the physiological signal and the generalizable intuitions of DTW. We also propose a dynamic template that will automatically adapt to new variations without domain expert intervention. Such automated comprehensive feature extraction can contribute to the waveform preprocessing steps required for prediction problems related to remote health monitoring by extracting actionable information from physiological signals collected by wearables.•We introduce Boosted-SpringDTW which simultaneously identifies all fiducial points of a given waveform stream.•We propose a probabilistic decision-making process that enhances DTW with minimal domain-specific heuristics, thus enabling the analysis of quasi-periodic signals.•We incorporate an automated dynamic template to adapt to evolving morphologies.Our contributions in this paper are summarized as follows:II.Boosted-SpringDTW – shown in A.Segmenting physiological signals into cardiac cycles involves assigning a probability, We detect the local maxima of a series of likelihood scores to identify the time steps that are the true cardiac cycle endpoints. Local maxima are defined by the dominant frequency, The search for true cardiac cycle endpoints occurs as DTW distances are computed. The process begins at the first potential B.We define the maximum number of templates allowed in the ensemble, III.th order Butterworth Bandpass Filter with cutoff frequencies of . We empirically determined Boosted-SpringDTW parameters We validate the effectiveness of Boosted-SpringDTW (single and dynamic template) to achieve comprehensive feature extraction from PPG waveforms that are impacted by respiration- and subject-induced morphological variations in the IEEE TBME Respiratory Rate Benchmark Data Set A.B.We evaluate the estimation of a well-studied physiological parameter from the detected fiducial points, IBI, which is highly regarded for health monitoring IV.Both versions of the proposed Boosted-SpringDTW framework show strong performance for identifying each class of fiducial points achieving scores over 0.949 for precision, recall, and F1. The improved F1 compared to the two baseline algorithms are the best reflection of overall performance since they represent the balance in TP, FP, and FN predictions. The dynamic template also yields an additional 1-2% improvement labelling SYS and MS points. Although the lowest RMSE values were by SpringDTW, it should be noted that RMSE reflects an average error of all predictions where this approach yielded far fewer positive predictions compared to each evaluated method for each fiducial point thus it was less susceptible to inherently noisy predictions.In The top performers for IBI estimation with each fiducial point are the two versions of Boosted-SpringDTW yielding MAE scores less than 12 ms and correlation coefficients greater than 0.98. We observe improved IBI estimation with SYS and MS points when Boosted-SpringDTW leverages the dynamic template, yielding a 28.5% improvement in MAE and a 2.0% improvement in the correlation coefficient values. In the difference plots shown in An immediate follow up work for this study would be to conduct rigorous stress testing to definitively evaluate the maximum levels of noise (such as motion noise) which Boosted-SpringDTW is able to handle. Also, extending the analysis to different modalities that may present new challenges would lead to further development of Boosted-SpringDTW capabilities. Furthermore, exploration into the value of this work to serve as a preprocessing tool contributing to end-to-end prediction frameworks. Particularly, Boosted-SpringDTW has the ability to segment and annotate physiological waveform segments that represent cardiac cycles which have been fed as input to prediction models pursuing tasks such as physiological parameter estimation V.In this work, we proposed Boosted-SpringDTW to perform comprehensive feature extraction for remote health monitoring. We enable the use of DTW for segmentation of quasi-periodic signals and without the need for pre-defined thresholds by combining the strengths of simple, minimal domain-specific heuristics and the generalizable DTW signal analysis method. We overcame the challenges associated with morphological variations in PPG with the notion of a dynamic template. We validated performance by evaluating precision, recall, F1-score, and RMSE performance when attempting to identify SYS, MS, and EP points of a collected PPG signal induced with variation in signal morphology due to inter-subject variability and respiratory behaviors. The proposed framework achieved superior performance for the fiducial point identification task compared to the original SpringDTW implementation and to the standard adaptive thresholding approach. This led to superior IBI estimation by Boosted-SpringDTW frameworks.


Reducing maternal mortality is a priority of Sustainable Development Goal 3.1 which requires frequent epidemiological analysis of trends and patterns of the causes of maternal deaths. We conducted two reproductive age mortality surveys to analyse the epidemiology of maternal mortality in Zimbabwe and analysed the changes in the causes of deaths between 2007-08 and 2018-19.n=10); an additional district selected from Harare. We calculated mortality incidence rates and incidence rate ratios per 10000 WRAs and pregnant women , in international classification of disease groups, using negative binomial models, and compared them between the two surveys. We also calculated maternal mortality ratios, per 100 000 live births, for selected causes of pregnancy-related deaths.We performed a before and after analysis of the causes of death among women of reproductive ages (WRAs) (12-49 years), and pregnant women from the two surveys implemented in 11 districts, selected using multi-stage cluster sampling from each province of Zimbabwe  in 2018-19.Zimbabwe experienced a decline in both direct and indirect causes of pregnancy-related deaths. Deaths from indirect causes declined mainly due to a reduction in HIV/AIDS-related and malaria mortality, while deaths from direct causes declined because of a reduction in obstetric haemorrhage and pregnancy-related infections. Ongoing interventions ought to improve the coverage and quality of maternal care in Zimbabwe, to further reduce deaths from direct causes.The online version contains supplementary material available at 10.1186/s12889-022-13321-7. Reducing maternal mortality is a priority of Sustainable Development Goal (SDG) 3.1 and the target is to reduce the global maternal mortality ratio (MMR) to 70 maternal deaths per 100 000 live births by 2030 and to leave no country with an MMR greater than double the global average . AnalysithEdition of the ICD (ICD-10) is used to classify diseases and causes of death in the general population, including women of reproductive ages (WRAs) . Ab. Ab42]. Zimbabwe halved deaths due to direct causes of maternal deaths  and the indirect causes of HIV/AIDS and malaria. Interventions implemented at various levels of the health system: policy development (roadmap), training (EmONC), providing access , monitoring and evaluation (MPDSR) achieved this impact. The achievements demonstrated how concerted multipronged interventions can reduce maternal mortality.Notwithstanding, direct causes continued to cause maternal deaths more than indirect, with a three-fold cause-specific MMR. Direct causes are continuing to contribute significantly to maternal deaths because of the ever-present threat of brain drain where skilled healthcare workers migrate to better-remunerating countries . ZimbabwAddressing obstetric haemorrhage would reduce deaths from direct causes by a third while addressing obstetric haemorrhage, abortion and hypertensive diseases would reduce deaths due to direct-causes by four-fifths. Thus, improving the coverage and quality of maternity care targeting these three causes remains a priority. Despite the presence of maternal waiting shelters in health facilities, women continue to experience significant first, second and third delays from various controllable factors, such as long distances to the nearest health facility and significant delays in getting transport to referral centres, coupled with human resource and commodity challenges at the referral centres. Efforts to reduce unskilled deliveries at home and ill-equipped primary-care facilities, improve emergency transport and increase access to the right care should continue –63. DiagThe limitation of this study is the possible under-representation of community deaths in 2018-19 data. The 2007-08 survey collected home deaths in the community, while the 2018-19 survey only included community deaths that were recorded in maternal death notification and CRVS records. Missing information for some deaths was addressed by triangulating the deaths across data sources. Deaths identified in CRVS records with incomplete information and not found in health records were classified as deaths of unknown causes, but these were few. Despite these limitations, the study identified sufficient deaths that were thoroughly reviewed by obstetricians to produce these findings.Mortality due to HIV and malaria has declined significantly in Zimbabwe in WRAs and pregnant women, though the two remain important causes of death. Zimbabwe has also significantly reduced pregnancy-related deaths from direct causes  through concerted multipronged interventions. Sustained investment into the health system focusing on improving coverage and quality of antenatal care and access to emergency obstetric care will further reduce deaths from direct causes . Efforts to contain the indirect causes of HIV and malaria should continue whilst increasing efforts to manage NCDs in pregnancy. The RAMOS should be repeated in the same districts before 2030, to assess progress towards the SDG target.Additional file 1. Additional file 2.


Emotional stress stemming from the complex grief associated with dementia care takes a greater toll on a family caregiver’s health than the physical effort of providing care. Finding Meaning and Hope, a 10-week workshop for caregivers of persons with dementia designed to promote understanding of grief and teach self-care concepts that mitigate stress. Each session features a video of a dementia care expert sharing strategies, followed by a facilitator-led discussion. Home practice activities help caregivers incorporate focal self-care strategies into their lives. This evaluation research assessed the association between workshop participation and caregiver outcomes in stress and well-being. 208 caregivers, 83 percent age 60 and older, completed the workshops between 2019 and 2021. Two standardized instruments were administered before and after workshop participation: the 12-item Caregiver Self-Assessment Questionnaire  and the 11-item Sense of Competence Questionnaire . Multivariate Linear Regression was used to assess factors associated with participant outcomes. Participation in more than half the sessions had a positive impact on stress management, emotional well-being, and coping, but fewer gains were made by caregivers with health limitations and those who began the workshops with lower emotional well-being . Given that family caregivers’ mental and emotional health has been negatively impacted by the COVID-19 pandemic , strategies to manage stress and bolster well-being are vital in enabling them to continue care provision when isolation may exacerbate the challenges they encounter. Finding Meaning and Hope offers a promising approach.


Cognitive reappraisal is an emotion regulation strategy that benefits health and well-being, but it is currently unclear why this relationship exists. The current study examined how adults react affectively to daily stressful events as a mediating pathway between cognitive reappraisal and health and well-being outcomes across a 20-year period. Participants  completed waves 1-3 of the Midlife in the United States survey series and the National Study of Daily Experiences, an 8-day daily diary. Results found that negative affective reactivity to daily stressors mediated the relationship between cognitive reappraisal and future health outcomes including the development of chronic conditions, functional limitations, and affective disorders. Exploratory analyses revealed that specific negative emotions, including being nervous and irritable explained this relationship. Findings from this study suggest that strengthening cognitive reappraisal skills may be a good way to reduce affective reactions to daily stressors and enhance long-term health and well-being outcomes.


JCI, Sena et al. developed a gene signature indicative of high AR activity to predict patient response to BAT, including a decline in both serum prostate-specific antigen (PSA) and tumor volume. Preclinical models showed that AR-mediated suppression of MYC, known to drive PC, was associated with decreased cell growth following SPA treatment. Because BAT eventually leads to resistance, the authors tested cycling between SPA and AR antagonism in a patient-derived xenograft and observed a delay in tumor growth. These findings represent a major step toward the informed use of BAT for advanced PC.Androgen deprivation therapy (ADT) is the longstanding treatment for advanced prostate cancer (PC) because androgen receptor (AR) is the key therapeutic vulnerability for this disease. Bipolar androgen therapy (BAT) — the rapid cycling of supraphysiologic androgen (SPA) and low serum testosterone levels — is an alternative concept, but not all patients respond and acquired resistance can occur. In this issue of the  Because androgen receptor (AR) signaling promotes growth and survival of prostate cancer (PC), blocking AR activation has been the cornerstone of treatment for more than 70 years. Androgen deprivation therapy (ADT) decreases the synthesis of the two major AR ligands, testosterone and dihydrotestosterone (DHT) using luteinizing hormone-releasing hormone (LHRH) agonists or LHRH antagonists. Despite initial therapeutic benefits, the disease inevitably progresses to an incurable stage termed castration-resistant PC (CRPC). This ultimate stage arises through a variety of mechanisms that promote persistent or reactivated AR signaling . This single arm, phase II clinical trial enrolled patients with metastatic CRPC whose cancer progressed on an ARSI and treated them with BAT for 12 weeks, followed by a combination treatment of BAT and the anti-PD1 agent nivolumab , which is highly expressed in PC. Further, a PDX that responded to SPA, as indicated by decreased tumor volume, showed decreased MYC. Similarly, patients who responded to BAT exhibited a larger decrease in MYC compared with nonresponders. Additionally, patients with decreased MYC also correlated with those that had higher AR activity before treatment . Althougpromotor .While SPA-mediated suppression of MYC is a leading proposed mechanism underlying BAT response, studies in preclinical models have implicated alternative mechanisms, including impaired DNA licensing, which is a process where high levels of ligand-stabilized AR bind to the origins of DNA replication and promote early arrest in S-phase . AdditioHSD3B1 leads to increased intratumoral DHT levels and promotes resistance to ADT and abiraterone treatment ) than men whose cancer had progressed on abiraterone  . Those tMW is a potential biomarker that may stratify responders from nonresponders; however, findings should be validated in an independent patient cohort. Although BAT therapy has the potential to improve the outcome of patients with metastatic CRPC while preserving good quality of life, treatment is not risk free and does not work for all patients. Therefore, BAT should not be used outside of a clinical trial, especially in combination with other therapies. Larger and randomized clinical studies are needed in the future to validate and determine the best way to integrate BAT into metastatic CRPC treatment algorithms.The Sena et al. study identified high AR activity as a requirement for clinical response to BAT, which is associated with downregulation of MYC. ARA


Long noncoding RNAs (lncRNAs) actively participate in breast cancer (BRCA) tumorigenesis via epigenetic mechanisms. Our study identified immune-related lncRNA (irlncRNA) pairs and compiled them into a set of noncoding gene signatures able to stratify subtypes of BRCA associated with variable degrees of survival and immune cell infiltration. A 40 immune-related lncRNA pair (IRLP) signature including 43 irlncRNAs was built, with high sensitivity and specificity for the prediction of survival in different molecular subtypes of BRCA. Results demonstrated that the low-risk group showed a significantly longer survival rate, and this novel IRLP signature was highly associated with survival status, T stage, metastatic disease, and overall stage in BRCA. Immune infiltrating analyses found that the low-risk group has a lower expression level of macrophage M2 and a higher expression level of immunosuppressed biomarkers than the high-risk group. DEirlncRNAs were further proven to be significantly related to the MAPK signaling, Jak-STAT signaling, and ErbB signaling pathways in BRCA. In conclusion, the 40 IRLP signature showed a promising clinical prediction value in the prognosis of different molecular subtypes and immunotherapy response in BRCA, and the underlying mechanism for these IRLPs warrants further investigations. Breast cancer ranks first for global cancer incidence in 110 countries in 2020 [Standard treatment of breast cancer relied heavily on a combination use of chemotherapeutics and hormonal disrupting agents in accordance with either their hormonal receptor  expression or their pathway-related molecular subtypes. In recent years, advanced translational research resulted in immune checkpoint inhibitors (ICIs) and modulators with some being investigated in breast cancer management. The contemporary guidance for drug usage depends on molecular diagnosis in monitoring gene signatures comprised of coding gene expression, mutational status, and DNA damage-related epigenetic phenomena. For example, high microsatellite instability (MSI-H), programmed death-1 (PD-1), programmed cell death ligand 1 (PD-L1), tumor mutational burden, and mismatch repair deficient (dMMR) have been widely used in clinic as predictive biomarkers for ICIs. With that, one may project the importance of gene modulators that modifies the immune cell niche as prognostic markers for BRCA.The immunotherapy field had achieved an immense breakthrough in the treatment of several cancer types . Even thlncRNAs are active participants in breast cancer tumorigenesis via epigenetic mechanisms, rendering them valuable predictive markers in this cancer progression and treatment. As a loose term for the entire collection of intracellular noncoding RNAs that were over 200 nucleotides, lncRNA demonstrates a myriad of actions implicated in regulating over 70% of coding gene expression through direct interactions or sponge effects . A subclIn our study, we intend to explore the predictive power of this overlooked collection of noncoding factors at the transcript level, particularly over how they may exert actions over immune surveillance, to reflect the likelihood of treatment responses and anticipate probable refractory cases. We sought to build an irlncRNAs-related computational model which efficiently predicts the immune landscape of breast cancer prognosis in different molecular subtypes. Our project also extended to investigate the functions of those with differential expression profiles across the subtypes.We downloaded the fragments per kilobase per million (FPKM) of normal tissue and breast cancer transcriptome profiling and metadata of diagnosed patients in these categories from The Cancer Genome Atlas (TCGA) database in May 2021 Table . Subsequp value < 0.001 with ir-mRNAs were distinguished as irlncRNAs, and the irlncRNAs with ∣log FC | >1.5 and FDR < 0.5 were identified as DEirlncRNAs. A 0 or 1 matrix was formed 0 as the expression of the former lncRNA in the DEirlncRNA pair of which its expression is lower than the latter, while 1 is indicative of the relative higher expression in the former. Qualified DEirlncRNA pairs shall hold a skewed proportion to having either 0 or 1 as higher than 20% in the matrix.Coexpression analyses were performed to identify the irlncRNAs. The lncRNAs that met the cut-off criteria of correlation coefficients > 0.4 and p < 0.05. Then, R package glmnet was performed for building the Lasso model with 10-fold cross-validation [i=1kβiSi.The valid DEirlncRNA pairs were screened using uni-Cox analysis; the threshold is lidation . A multiOur study also calculated the AUC values of each model, and R package ROCR was used for generating the receiver operating characteristic (ROC) curve plots of 1, 3, and 5 years . Then, wt-test is performed between the signature and clinicopathological features. Survival status, age, gender, metastatic disease, N stage, T stage, and clinical stage were considered variables. To further verify if the RS can serve as an individual predictor, uni-Cox and multi-Cox were conducted between the high- and low-RS groups.To confirm the clinical value of our novel model, the paired BRCA is usually divided into 4 molecular subtypes—Her2-enriched, luminal A, luminal B, and triple-negative breast cancer (TNBC). For patients without the metadata of intrinsic molecular subtypes, PAM50 function was used for classifying the molecular subtypes . The surTo infer the possible biological functional pathway of irlncRNAs in the prognostic signature, RNA central  was usedp value < 0.05 was considered significant in our study, where visualization was labelled as below: ∗∗∗<0.001, ∗∗∗<0.01, and ∗<0.05.The R package ggplot2 and ggpubr package was performed to produce the plots. A Firstly, we obtained the transcriptomic profiles of 113 normal and 1109 tumor cases from TCGA database. Those data were annotated with gene transfer format (GTF) files downloaded from the Ensembl database . In furt63 DEirlncRNAs produced 1011 valid DEirlncRNA pairs. Then, 40 DEirlncRNA pairs were screened by Lasso regression analysis from 1011 valid DEirlncRNA pairs  for signp < 0.001) . Those identified genes were further subject to GO enrichment analysis  and head and neck cancer niches, T cell-derived immune privileges demonstrate worsen prognosis along with immunotherapeutic regimes. The proven hardships in the application of ICIs in breast cancer types had dwindled continuous research interest in immunotherapy. Furthermore, coupled with a lack of an effective immune gene signature for the predictive outcome, the apparent pharmacological idiosyncrasy further hampered the development of personalized regimes as the last resort in breast cancer treatment. Indeed, in ICI therapy as in all other personalized treatment, precision medicine approaches by molecular profiling remains the key to improving all therapeutic success. As a result, it must be emphasized that the medical status quo might have indirectly caused an underaddressed call in the use of ICIs in combating the disease, particularly those highly immune infiltrated subtypes per se.The evaluation of the expression level of PD-1/PD-L1 and the presence and activation status of tumor-infiltrating T cells are promising tools to improve the effect of tumor immunotherapy , 19. HowNevertheless, lncRNAs were implicated in driving malignant phenotypes in almost all walks of cancer types, and many such transcripts formed the basis for prognostic expression signatures of BRCA with merit success. For example, Shen et al. identified 11 lncRNAs as a prognostic signature for BRCA , and Ma Indeed, there were past attempts to devise expression profiles for constructing irlncRNA prognostic signatures for breast cancer. However, previous signatures are mostly based on quantifying transcript expression levels and seemingly ignored the high difference between heterogeneous biological and clinical features in molecular subtypes. Moreover, in coding genes categorized by the molecular pathway, they partake in; the biological function of those gene modulators could be revealed from how they interact with their binding partners directly. Unlike those coding gene products, most lncRNA driven by its mode of action as antisense transcripts tend to bind multiple target mRNAs while its presence may lead to variant expression changes of the binding partner through both direct and indirect interactions.A highlight in the current study is an emphasis on filtering out only reasonable signatures based on irlncRNA pairings and discriminating the unified or divergent function of these enriched gene sets across different molecular subtypes. It remains difficult to single out a precise molecular action for any lncRNA merely by its own expression, and therefore, we sought to rely on pinpointed coexpression pairs. Their largely uncharted molecular functions also prompted us to explore the stage-specific actions of our clustered DEirlncRNA pairs along with the immune feature-stratified disease subtypes.lncRNA pairs are a reasonable optimized method for building a prognosis signature compared to single lncRNAs. lncRNA pairs combined with higher or lower expression lncRNAs ignore their exact expression levels in the signature, which renders detecting specific expression values of all irlncRNA in the expression profile unnecessary. Indeed, such lncRNA pair signature had shown selectively greater advantage than the transcriptome approach in predictive performance in various cancers , 10. TheSome DEirlncRNAs in the 40 IRLP signature have been validated to be involved in the occurrence and development of tumors. lncZEB2-AS1 and MEF2C-AS1 have been demonstrated as poor biomarkers for patients with BRCA , 25. Our+ T cells correlated with a better response to traditional chemotherapy and ICIs in patients with TNBC [In this study, our IRLP signature can reflect the characteristics of the TIME based on the tumor-infiltrating lymphocytes which were adopted to define patients with BRCA as most likely to benefit from ICI therapies. We investigate whether RS was related to the TIME including TIICs, checkpoint-related biomarkers, and immunosuppression-related signal pathways. Research has demonstrated a higher infiltration level of CD8ith TNBC , 30.+ T cells as well as higher expression levels of checkpoint-related biomarkers. Our results indicate that this novel IRLP signature could preciously classify the patients who can benefit from giving any sort of immunotherapy.By integrating these statistical results, data demonstrated that the low-risk group classified by the IRLP signature was positively associated with a higher infiltration level of CD8Furthermore, the macrophage M2 phenotype follows an immunosuppressive action in general to promote tumor growth. As such, tumors highly infiltrated with macrophage M2 cell  and low The current prediction model had not been implemented in a large patient cohort, and therefore, the predictive power for our proposed gene panel might be overestimated in this molecular-divergent BRCA sample pool. Secondly, the coexpression model relied on carcinoma in situ samples, and this limited the usage of our signature panel aside from conducting a biopsy. In fact, lncRNAs were found to exist in exosomes and also freely circulating form in the bloodstream; therefore, the utility of our panel as a surrogate marker could be expanded if being considered in a liquid biopsy setting.Finally, we had yet to demonstrate the specificity of our gene signature across different diseases, which might or might not hasten its clinical application as a surrogate biomarker for prognosis in all immune-privileged primary cancer lesions. As much as we would like to develop a specific panel sufficient but limited to stratify BRCA subtypes in the assessment of the suitability of ICIs, there remains a high chance that the same noncoding genes may be useful to all these cancer types towards a unified approach.This study successfully constructed an IRLP signature for the predictive prognosis of BRCA patients independent of an individual lncRNA expression. Moreover, our signature reliably predicts individuals who will benefit from the immunotherapy, thus contributing to the development of personalized immunotherapy treatment for BRCA patients.


We provide evidenceof a local synaptic nanoenvironment in thebrain extracellular space (ECS) lying within 500 nm of postsynapticdensities. To reveal this brain compartment, we developed a correlativeimaging approach dedicated to thick brain tissue based on single-particletracking of individual fluorescent single wall carbon nanotubes (SWCNTs)in living samples and on speckle-based HiLo microscopy of synapticlabels. We show that the extracellular space around synapses bearsspecific properties in terms of morphology at the nanoscale and innerdiffusivity. We finally show that the ECS juxta-synaptic region changesits diffusion parameters in response to neuronal activity, indicatingthat this nanoenvironment might play a role in the regulation of brainactivity. In particular, the design and combinationof experimental strategies offering nanoscale resolution and sectioningcapabilities are needed to correlate the narrow and tortuous environmentwith specific cellular structures.3Neuronal communication in thecentral nervous system mainly occurs at the level of synapses throughthe release of neurotransmitters in the synaptic cleft and the activationof postsynaptic receptors. Neurotransmitters can spill over from synapsesand act at a distance through a process known as “volume transmission”,in which signaling molecules navigate within the brain extracellularspace (ECS).6 Compared with free diffusion in an “open space”where molecules move randomly, diffusion in the ECS is criticallydependent on the physical and chemical structure of the local microenvironment.7 Zheng et al. investigated the extracellular diffusivityinside the cleft of synapses formed by hippocampal mossy fibers whichcould be resolved by diffraction limited microscopy approaches, suggestingreduced diffusion when compared to free medium.8 Yet, understanding the mechanisms through which the ECSmodulates neuronal communication, particularly around excitatory synapsesat the nanoscale, still represents a key challenge in brain research.3 Because the characteristics of the ECS aroundsynapses remain unknown at the submicron scale in native live braintissue, it is important to establish whether the synaptic ECS environmentdisplays specific dimensions and diffusional properties.Changes in the ECS can affect neuronal excitability and signaltransmission by altering local ion concentrations in the healthy anddiseased brain.Totackle this question, we developed a correlative imaging approachbased on single-particle tracking of individual fluorescent singlewall carbon nanotubes (SWCNTs) in living brain tissues and on speckle-basedmicroscopy of synaptic labels 1A. The f9 dueto their unique brightness, photostability, and near-infrared emissionrange in the biological window.10 SWCNTsalso demonstrated the capability to locally probe in situ chemical species, including neurotransmitters.11 At the single nanotube level, we have recently shown thatSWCNTs can access ECS in the brain tissue of young12 and adult13 rodents, and theirdiffusion trajectories can reveal ECS local dimensions at the nanoscale.It is noteworthy that another optical approach based on STED microscopy14 could provide similar structural ECS dimensions.Using these optical methodologies, ECS remodeling in a neuropathologicalcondition was successfully reported.15 Furthermore,contrary to other microscopy techniques displaying nanoscale resolutionfor the study of structural tissue features , SWCNT tracking allows us to investigate ECS architecturesin intact living brain at unrivaled depths (>10 μm), andinaddition, it has the unique ability to reveal diffusion propertiesinside the tissues. To locally probe the ECS dimension and diffusivityaround synapses deep inside living brain tissues, the use of SWCNTsthus emerged as the tool of choice.Near-infrared luminescent SWCNTs are interesting nanoparticlesfor deep brain tissue microscopy,16 HiLo is a wide-field fluorescence microscopymethod which provides excellent optical sectioning capabilities inthick biological samples, akin to confocal microscopy but at higherimaging rates and with simpler instrumentation. It requires the acquisitionof two images: one using a uniform illumination and one with structuredillumination (here the illumination transported through a multimodefiber is randomly structured by the speckle). These two images areused to extract the high and low frequency in-focus contents , leading to a full resolution in-focus imagecontaining the entire frequency bandwidth of the imaging system . We focused on  SWCNTsemitting at 985 nm which are efficiently excited at 845 nm while minimizinglight absorption by the tissue.19 Bright SWCNTs were sparsely and individually detected at high signal-to-noiseratio with low autofluorescence . Importantly, the SWCNThigh aspect ratio and intrinsic rigidity play a decisive role here,slowing down nanotube diffusion in the ECS maze while ensuring highaccessibility to nanoscale environments.20 These are unique features of these bright non-photobleaching 1Dnanoparticles.Biocompatible fluorescent SWCNTs were prepared by encapsulationwith phospholipid–polyethylene glycol (PL–PEG) molecules.This coating minimizes nonspecific adsorption onto biological structuresanotubes 1D, which21 the measured nanotube length distribution was centeredaround ∼600 nm  and nanotube length (long axis of the Gaussian fit). Taking intoaccount the exciton diffusion range that decreases the apparent nanotubelength in fluorescence images due to end quenching, ∼600 nm . Thisna22). An image correlation analysiswith SWCNT localizations allowed us to create a distance-to-synapseinvestigation of the ECS features. We first analyzed SWCNT local diffusivityin the different coronal areas. Local diffusivity was defined as thenormalized local instantaneous diffusion along trajectories. For this,the two-dimensional mean-squared displacement (MSD) was calculatedas a function of time intervals along a sliding window of 390 ms foreach trajectory. By approximating the MSD as linear at short timeintervals, a linear fit of the first 90 ms yielded Dinst, the instantaneous diffusion coefficient. The normalizeddiffusivity was then obtained by calculating Dinst/Dref where Dref is the calculated diffusion coefficient of carbonnanotubes freely diffusing in a fluid having the viscosity ηref of the cerebrospinal fluid (CSF)7 .In order to explore theECS environment around the synapses, theregion around each GFP-PSD95 centroid was segmented by a series ofconcentric coronal areas of 100 nm widths 2A. A maxMaterial and Methods and Figure S4A,B. Furthermore, usingthe experimental SWCNT trajectories, we also generated “fake”(randomized) localizations of synapses in regions where no positiveGFP-PSD95 signals were experimentally identified: no specific (enhanced)diffusivities were subsequently generated around the “fake”synaptic areas (10 SWCNT.mL–1) far exceeding those observed in our brain slices to ensure thatthe vast majority of synapses were statistically exposed to a SWCNT.We report that the presence of SWCNTs, for tens of minutes, do notalter the frequency of synaptic transmission in active hippocampalneuronal network , where the shorterdimension represents the local ECS dimensions (ξ). Spatial mapsof local diffusivities (Dinst/Dref) and ECS local dimensions were thus generated axis of the microscope,such that juxta-synaptic regions might also be contaminated by non-juxta-synapticfeatures. In any case, we found that the juxta-synaptic diffusivityis 6-fold faster than the non-juxta-synaptic region , we next ran an extensive characterizationof the ECS features pooled into these regions 3. In addenerated3A, reveajuxta = 193 nm – median_non-juxta = 83 nm; p < 0.001; p < 0.01).r = 0.138; i) the diffusivity of SWCNTs is mainly influenced by the molecularcomposition of the space, and (ii) spatial constrictionsof cellular walls are not necessarily the central determinant of ECSdiffusion inhomogeneities at the nanoscale near synapses.In general,the broad shape of the cumulative distributions confirmedthat the ECS is a highly heterogeneous milieu, where diverse localproperties can impose a wide range of diffusivity and dimensionalvalues. Indeed, in contrast to a free medium, diffusion in the brainECS can be hindered by cell processes, astroglia, macromolecules ofthe matrix, wall drag, and the presence of charged molecules. In thisenvironment, the diffusivity is also dependent on the hydrodynamicdimension of the diffusing probe, resulting in lower diffusivitiesfor larger objects. Interestingly, median values of diffusivity andlocal dimensions evaluated at the level of individual juxta-synapticregions are uncorrelated or decrease  neuronal activity . This observation suggests that the neuronalnetwork can accommodate to neuronal activity changes (either increaseor blockade) through ECS regulation. Finally, We nextfocus on the juxta-synaptic region to compare the differentconditions 5. SWCNT 23 Thesechanges were related to cell swelling, cell loss, astrogliosis, rearrangementof neuronal and astrocytic processes, and changes in the extracellularmatrix. Plastic changes in ECS volume, tortuosity, and anisotropycan also affect the communicaton between neurons and other cell types.24 Here, TTX-treated samples have slower diffusivitythan the ones incubated with BIC (p < 0.001),but the local dimensions remain comparable between conditions, suggestingthat the differences between BIC and TTX relies on the chemical modificationsof the juxta-synaptic region. This is further supported by the correlationanalysis evaluated for individual GFP-PSD95 positive clusters, whichrevealed a higher correlation between local diffusivity and dimensionsin the juxta-synaptic region for BIC-treated with respect to TTX-treatedsamples (A significant and global decrease in the ECS volumefraction andan increase in diffusion barriers have been reported during neuronalactivity and pathological states.samples .Altogether,our study revealed the existence of a juxta-synapticECS nanoenvironment within 500 nm from excitatory synapses in hippocampalbrain slices, not accessible with previous approaches due to limitedresolution. This observation was possible by correlating the dynamicsand superlocalization of NIR-emitting carbon nanotube with HiLo microscopyof labeled synapses in live brain slices. Increasing or decreasingsynaptic activity specifically modified the ECS diffusion and morphologicalparameters in the juxta-synaptic region. Such regulation of the ECSnanoenvironment around synapses would strongly influence the diffusionof neurotransmitters and modulators in the brain tissue, impactingneuronal network physiology and pathology.


Serving as the metabolic hub of the human body, the liver is a vital organ that performs a variety of important physiological functions. Although known for its regenerative potential, it remains vulnerable to a variety of diseases. Despite decades of research, liver disease remains a leading cause of mortality in the United States with a multibillion-dollar-per-year economic burden. Prior research with model systems, such as primary hepatocytes and murine models, has provided many important discoveries. However, progress has been impaired by numerous obstacles associated with these models. In recent years, induced pluripotent stem cell (iPSC)-based systems have emerged as advantageous platforms for studying liver disease. Benefits, including preserved differentiation and physiological function, amenability to genetic manipulation via tools such as CRISPR/Cas9, and availability for high-throughput screening, make these systems increasingly attractive for both mechanistic studies of disease and the identification of novel therapeutics. Although limitations exist, recent studies have made progress in ameliorating these issues. In this review, we discuss recent advancements in iPSC-based models of liver disease, including improvements in model system construction as well as the use of high-throughput screens for genetic studies and drug discovery. Liver disease contributes to tens of thousands of deaths each year in the United States alone ,2. The mPrimary hepatocytes taken from living or recently deceased patients have classically been used for studying liver disease in vitro. As these cells are genetically identical to the donor, they allow for the identification and study of genes and mutations relevant to the patient’s specific pathology. However, primary hepatocytes rapidly de-differentiate when cultured, losing their native physiology, rendering long-term functional studies difficult . LimitedImmortalized human liver cell lines provide a means of overcoming the insufficient supply of primary cells for large-scale studies. These cells can be maintained and expanded easily, making them amenable for high-throughput screening. However, these cells also show significantly altered metabolism associated with their malignant genotypes ,6. For eRodent models have been highly useful for both drug discovery and modeling human disease. In liver research, these systems provide an advantage over primary hepatocytes in terms of availability and amenability to genetic manipulation, especially with the rise of genomic editing tools, such as the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system . They alInduced pluripotent stem cells (iPSCs) are a relatively new technology that can address the limitations of other model systems. iPSCs can be generated from a patient’s somatic cells via the induction of certain transcription factors—namely c-Myc, Klf4, Oct3/4, and Sox2 . These riPSCs can be differentiated to hepatocyte-like cells over a 20-day period using recombinant growth factors and culture conditions that mimic those experienced by hepatocyte precursors in the developing human fetus ,19. AlthAs an alternative to the use of growth factors, small molecule regimens have been developed for the generation of HLCs from iPSCs. These regimens typically involve three phases, for example using Wnt/β-catenin activators to produce endoderm, TGF-β inhibitors to define the hepatic fate, and a small molecule/glucocorticoid mixture to drive hepatocyte maturation and proliferation . The resWhile iPSC-derived hepatocytes show numerous advantages as a model system for liver disease, they are not without their limitations. Chief among these is the well-documented difference in Phase I and II enzyme expression between iPSC-derived hepatocytes and mature primary hepatocytes ,27,28,29In the liver, hepatocytes perform different physiological processes based on their three-dimensional arrangement in lobular units. These functional differences are largely regulated by cell–cell and cell–extracellular matrix (ECM) interactions based on the hepatocyte’s position in the lobule . MonolayOther techniques involve the construction of three-dimensional scaffold matrices that can then be populated with cells. HLCs grown on these scaffolds demonstrate higher expression of CYP family genes and decreased expression of fetal hepatocyte markers when compared with 2D-cultured HLCs, indicating a better maturation more suited for toxicology studies ,40,41. TAdvancements in HLC differentiation and modeling have also proven useful in studying infectious diseases such as malaria and hepatitis B. An iPSC-based model of malaria was improved by using a small molecule to induce CYP expression such that antimalarial drugs requiring CYP bioactivation could be effectively tested . CocultuTRIB1 gene and found a lipid metabolism phenotype similar to that seen in a TRIB1-deficient mouse model, supporting a functional role for TRIB1 in humans [GPC1 and ADD3, which had been associated with biliary atresia in a prior GWAS [iPSC-derived HLCs can be used effectively for high-throughput studies of gene function. Genome-wide association studies (GWAS) have facilitated the identification of many genes potentially involved in a variety of pathologies. However, the correlative nature of these studies requires that further validation of identified candidates be performed. iPSC-based models are a viable approach for further exploring the contributions of allelic variations to liver disease. Abbey et al., 2020 used an iPSC-derived HLC organoid model to investigate the GWAS-identified n humans . Tian etior GWAS . iPSC-baior GWAS ,53. Diseior GWAS  and famiior GWAS , providiThe amenability of iPSCs to genetic manipulation via tools such as CRIPSR/Cas9 make these systems attractive for modeling and correcting liver diseases with genetic etiologies. Jing et al., 2018 created a model of mitochondrial DNA (mtDNA) depletion syndrome using CRISPR to knock out deoxyguanosine kinase in iPSCs, recapitulating the decreased mtDNA copy number and impairing the mitochondrial function seen in mtDNA depletion syndrome patients . CRISPR-As previously stated, iPSCs show great promise for modeling genetic disorders and can faithfully recapitulate pathologies seen in patients. Familial hypercholesterolemia is an autosomal dominant disorder associated with deficiency in the low-density lipoprotein receptor (LDLR). The absence or loss of function in this receptor leads to profoundly increased lipoprotein secretion by the liver in addition to impaired clearance of LDL from the blood. This dysregulation vastly increases blood lipoprotein levels, leading to premature and severe adverse cardiovascular effects . Cayo etIn addition to genetic disorders, environmental pathologies can also be modeled using iPSC-derived hepatocytes. Tian et al., 2016 exposed mature HLCs to pathological concentrations of alcohol and observed a dose-dependent increase in markers associated with mitochondrial DNA damage, lipid accumulation, and hepatocellular carcinoma. Strikingly, immature HLCs that more closely modeled human fetal hepatocytes did not show this increase in pathological markers although there was a significant reduction in proliferating cells . StudiesIn addition to mechanistic studies of liver disease, iPSC-based models can be used for high-throughput assays to discover and develop novel therapeutics. Jing et al., 2018 conducted a small molecule screen using their model of mtDNA depletion syndrome, resulting in the identification of NAD+ as a potential treatment . Cayo etThe suboptimal expression of xenobiotic enzymes in iPSC-derived HLCs have historically hampered their use in toxicity studies. However, multiple groups have recently developed iPSC-based systems with improved expression profiles for use in toxicity screening. These systems possess unique designs and conditions, but each demonstrate results comparable to those obtained from studies with primary hepatocytes ,101,102.iPSC-derived hepatocyte-like cells are an extremely useful technology for the study of liver disease. Although HLC-based models are not without their constraints, advancements are continuing to be made in improving their functional relevance and increasing their scalability. The number of diseases that can be successfully modeled with HLCs is continually expanding, opening the door for a deeper understanding of these diseases and the discovery of new treatments for them. Improvements in HLC-based toxicity studies raise the prospect of eventually tailoring drug therapy to individual patients based on data generated from cells sharing their exact genotypes. Additionally, the development of more disease-specific iPSC panels will allow for the identification of potential therapeutic targets and novel therapeutics for both common and rare liver diseases.