Patients with end-stage kidney disease (ESKD) have a high prevalence of cardiovascular disease; it is the leading cause of death in these patients and the optimisation of their cardiovascular health may improve their post-transplant outcomes. Patients awaiting renal transplant often spend significant amounts of time on the waiting list allowing for the assessment and optimisation of their cardiovascular system. Coronary artery disease (CAD) is commonly seen in these patients and we explore the possible functional and anatomical investigations that can help assess and manage CAD in renal transplant candidates. We also discuss other aspects of cardiovascular assessment and management including arrhythmias, impaired ventricular function, valvular disease, lifestyle and pulmonary arterial hypertension. We hope that this review can form a basis for centres hoping to implement an enhanced recovery after surgery (ERAS) protocol for renal transplantation. Cardiovascular disease (CVD) is the leading cause of morbidity and mortality among those with end-stage kidney disease (ESKD)  and kidnThe burden of cardiovascular disease will continue to increase as candidates on the kidney transplant waiting lists get older and more comorbid. In 2011, 62% of kidney transplantation candidates were over 50 years of age compared with 28.7% of kidney transplantation candidates in 1991 .The imbalance between the availability of organs and the number of patients on the transplant waiting list necessitates objective selection criteria to guide transplant candidacy decisions. Preoperative cardiovascular assessment can be utilized to identify suitable candidates who are at a lower risk of adverse events and guide management to optimise transplantation outcomes.Our review aims to highlight important points for a clinician to consider while performing a comprehensive, holistic cardiovascular screening assessment starting from the bedside assessment to the utilization of novel, highly sophisticated screening methods such as cardiac positron emission tomography (PET). We aim to provide a narrative review on the cardiovascular assessment and management of CAD, arrhythmias, heart failure, pulmonary hypertension, valvulopathies and lifestyle factors. This is however a very broad topic and we are not able to cover every issue relating to the cardiovascular system. Other complex issues including hypertension, right ventricular overload syndrome and hemodynamic monitoring are not covered in this review. We do include a summary of the most recent 2020 Kidney Disease: Improving Global Outcomes (KDIGO) Clinical Practice Guideline  and disc2, the probability of developing CAD increases linearly  ,96 95,96Similar to findings in non-CKD patients, pharmacological rhythm control and rate control strategies are equivalent in their efficacy in terms of risks of heart failure, stroke and mortality. Catheter ablation is superior to pharmacological antiarrhythmic therapies in achieving freedom from AF recurrence . The utiImpaired ejection fraction (EF) is known to be a poor prognostic marker in heart failure with those with EF < 30% on HD having a nine times higher risk of mortality than those with EF > 60%. Multiple medical therapies including beta-adrenoreceptor antagonists (beta-blockers), angiotensin-converting enzyme inhibitors (ACEi), mineralocorticoid receptor antagonists and neprilysin inhibitors are known to reduce mortality in certain populations with heart failure. These medications should be optimised by cardiologists in patients with heart failure on the renal transplant waiting list. In those with symptoms of heart failure or a known diagnosis of heart failure, it is reasonable to recommend regular echocardiograms to monitor the ejection fraction. Arguably, the main role of these screening echocardiograms is to aid with the allocation of resources; given the high mortality of those with EF < 30%. Patients with ESKD are known to have a high incidence of valvular disease with valvular calcification being reported in more than 50% of patients in some studies ,104. We There is a multitude of evidence describing the criteria for medical or surgical intervention in these valvulopathies as well as good RCT evidence of their survival benefit over medical management. As patients are often on the waiting list for many months or years, the patient may benefit from these procedures as they primarily reduce the risk of cardiac events. There is also evidence to show that patients with these valve disorders have worse perioperative outcomes in non-cardiac surgery and a recent retrospective study showed that an aortic valve replacement had better perioperative outcomes than those with severe AS but no replacement ,108,109.p < 0.001) in one study [Cigarette smoking is known to worsen peri-operative outcomes with an increased incidence of primarily wound and pulmonary complications ,115,116.ne study . A cohorne study . Most prne study ,123.Possible interventions in this area range from behavioural to pharmacological. Two particularly intensive studies including weekly interventions and counselling sessions had a large effect on smoking cessation at the time of surgery (RR 10.76 95% CI 4.55–25.46) ,124,125.Obesity is associated with worse outcomes post-operatively with generally increased rates of wound complications and specifically graft loss and death in kidney transplantation ,128. As There is also much interest in the possibility of bariatric surgery in either potential renal transplant candidates or post-renal transplant ,138,139.Pulmonary arterial hypertension (PAH) is becoming increasingly recognised in patients with ESKD with some studies reporting an incidence of 32% . It is kPAH is defined by a mean pulmonary artery pressure (PAP) of greater than 25 mmHg on right heart catheterisation (RHC). This is however an invasive procedure so TTE to measure estimated pulmonary artery systolic pressure (PASP) is commonly used as an initial technique to assess pulmonary pressures. Echocardiographic measurements have a sensitivity of 83% and specificity of 72% in detecting PAH so patients with an elevated PASP should then go on to have an RHC to confirm the presence of PAH .Intrinsic arteriopathy due results in increased pulmonary vascular resistance : this caLeft ventricular dysfunction ,144: LV Hypoxic pulmonary vasoconstriction : pulmonaChronic thromboembolic pulmonary hypertension (CTEPH) : this isMultifactorial or unclear aetiology: these patients have PAH due to haematological disorders , systemic disease (e.g Langerhans cell histiocytosis), metabolic disease or even unknown aetiology. In ESKD, a potential culprit could be a high-output arterio-venous fistula . RetrospIt has variable aetiologies which are important to differentiate to allow for optimal management. The WHO classification has five categories:The aetiology of PAH can be established by patient history (OSA), blood investigation , pulmonary angiogram (CTEPH), TTE  and RHC. RHC can be particularly useful in determining pulmonary capillary wedge pressure (PCWP), pulmonary vascular resistance (PVR) and cardiac output. With these parameters, it is possible to distinguish between group 1, group 2 and group 5 PAH and treat accordingly. Given the high prevalence of PAH and the fact that patients on the renal transplant waiting list are already screened with TTE, we believe that further screening with RHC in those with elevated PASP to diagnose and categorise PAH is warranted in patients on the kidney transplant waiting list. This can then be managed according to subtype with further studies needed to confirm whether treatment improves operative outcomes for patients.A summary of some considerations in the screening of renal transplant candidates is included in Cardiovascular disease is a significant cause of morbidity and mortality in patients with ESKD but these patients are commonly excluded from major cardiovascular clinical trials. There remain many unanswered questions on optimal assessment and management of cardiovascular disease in patients with renal dysfunction. More randomized control trials incorporating patients with CKD and ESKD may benecessary.There is an expansion of non-invasive assessment of the coronary artery architecture with a refinement of the criteria for intervention but more research is needed to guide decisions about suitable tests based on baseline cardiovascular risk and renal function. Some of these tools, including CMR and PET imaging, are yet to be thoroughly evaluated in patients with CKD and ESKD and the sensitivity and specificity of these tests in this patient population are yet to be identified. The optimal frequency of CAD screening while on the transplant waiting list is unknown, and this question is currently being investigated by the CARSK  trial. Whether CAD screening leads to improved post-transplant outcomes is also unproven. Prospective randomized trials are needed to define which subsets of patients might benefit from pretransplant intensified medical management or from revascularisation.Pre-transplant cardiac arrhythmias are associated with adverse outcomes but currently only a baseline ECG is recommended to evaluate for arrhythmias pre-operatively. Studies looking at the utility of ambulatory rhythm monitoring and other strategies to identify underlying arrhythmias would be beneficial. There is a lack of evidence from RCTs on the optimum dosing regimen, efficacy and safety of anticoagulants in advanced CKD. With structural heart disease, percutaneous options are becoming more widespread and further prospective studies are needed to study their benefit in the ESKD population and whether these improve post-transplant outcomes.In conclusion, we hope that our review adds to the very complex issue of optimisation of the cardiovascular assessment and management of renal transplant candidates (summarized in


Freezing of gait (FOG) is a particularly debilitating motor deficit seen in a subset of Parkinson’s disease (PD) patients that is poorly responsive to standard levodopa therapy or deep brain stimulation (DBS) of established PD targets such as the subthalamic nucleus and the globus pallidus interna. The proposal of a DBS target in the midbrain, known as the pedunculopontine nucleus (PPN) to address FOG, was based on its observed pathology in PD and its hypothesized involvement in locomotor control as a part of the mesencephalic locomotor region, a functionally defined area of the midbrain that elicits locomotion in both intact animals and decerebrate animal preparations with electrical stimulation. Initial reports of PPN DBS were met with much enthusiasm; however, subsequent studies produced mixed results, and recent meta-analysis results have been far less convincing than initially expected. A closer review of the extensive mesencephalic locomotor region (MLR) preclinical literature, including recent optogenetics studies, strongly suggests that the closely related cuneiform nucleus (CnF), just dorsal to the PPN, may be a superior target to promote gait initiation.We will conduct a prospective, open-label, single-arm pilot study to assess safety and feasibility of CnF DBS in PD patients with levodopa-refractory FOG. Four patients will receive CnF DBS and have gait assessments with and without DBS during a 6-month follow-up.This paper presents the study design and rationale for a pilot study investigating a novel DBS target for gait dysfunction, including targeting considerations. This pilot study is intended to support future larger scale clinical trials investigating this target.ClinicalTrials.gov identifier: NCT04218526  Gait dysfunction is a common and heterogeneous feature of Parkinson’s disease (PD), significantly impacting patients’ quality of life and increasing their risk for falls . Common FOG is considered the most disabling of these gait deficits and is described as the transient and episodic failure to move forwards, despite intending to walk . PatientThe pathophysiology of FOG is incompletely understood, but observations that it can be refractory to dopaminergic therapy suggest that it involves both dopaminergic and non-dopaminergic mechanisms . FOG is The mesencephalic locomotor region (MLR) is a physiologically defined area in the midbrain tegmentum, where electrical stimulation was found to initiate locomotion in cats , 17. SinOver the past 15 years, several centers have reported on DBS of the pedunculopontine nucleus (PPN), a putative anatomical component of the MLR, for postural instability and gait dysfunction in PD . DespiteSeveral reasons have been posited to explain this lack of efficacy, including potential species differences in MLR function, and the degeneration of MLR neurons in PD –42. A reThus, this pilot study is designed to test the safety, feasibility, and preliminary efficacy of CnF DBS in alleviating FOG. Through this and future larger scale studies, we ultimately aim to explore the hypothesis that the CnF represents the neuroanatomic basis of the MLR and that suboptimal targeting may have played a role in the equivocal results of prior PPN DBS studies. We hope to confirm the findings in Goetz et al. , which sClinicalTrials.gov: (NCT04218526). The study outline is shown in Fig. This study is a prospective, non-randomized, single-arm, open-label, pilot clinical trial designed to evaluate the safety and feasibility of delivering DBS to the CnF to alleviate freezing of gait in PD patients with severe, levodopa-resistant gait freezing. This study was approved by the University of Miami Human Subject Research Office (UM HSRO) and the US Food and Drug Administration with an Investigational Device Exemption as a phase I trial. The trial is registered in ov: NCT0418526. ThSuitable eligible patients will be screened for study participation and begin the consenting process, where the study objectives and the risks and benefits of participating are explained to them. Informed consent forms for the study will be filled out by subjects to confirm enrollment and they will undergo a baseline assessment, including a thorough neurological and neuropsychiatric examination, as well as standardized subjective and objective gait assessments. The Columbia Suicide Severity Rating Scale is included during screening given the known risk of suicidality with DBS for PD . After eWe are actively screening and recruiting patients for this study from the Movement Disorders Clinic at the University of Miami Hospital. As this is a pilot study, we plan to enroll 4 patients with PD with levodopa-resistant FOG. A movement disorder neurologist will assess the eligibility of a patient based on the inclusion and exclusion criteria are detailed below.Age 40–75FOG is the primary complaint despite optimized dopaminergic therapy by a movement disorder neurologistClinical observation of FOG in the ON/OFF states by a movement disorder neurologistPD stage 3 with good response to levodopa (defined as greater than 20% improvement in MDS-UPDRS score), except for severe gait disorderPD stage 3 with severe gait dysfunction and predominant axial symptoms defined as TD/PIGD ratio ≤ 0.90  [Minimal tremor, bradykinesia, and rigidity, or well controlled with levodopa.Agrees to full 6-month study participationIndividuals with major executive dysfunction, dementia , depression (Beck Depression Inventory II ≥ 25), or other neurocognitive impairmentsPresence of major medical co-morbidities and other surgical contra-indicationsIndividuals requiring diathermy, transcranial magnetic stimulation, or electroconvulsive therapyIndividuals with prior intracranial surgeryIndividuals with non-MRI compatible metallic implants in their head or active implantable devices anywhere in the bodyIndividuals who are pregnant, breastfeeding, or the desire to become pregnant during the studyIndividuals on investigational drugs or any other intervention (not part of the guidelines for management of PD) known to have a potential impact on outcomeHistory and physical exam, including a neurological and clinical gait assessmentBlood pressure and postural dropPre-operative lab work, including 12-lead electrocardiogram and chest x-rayNeuropsychiatric evaluation, including the Mattis Dementia Rating Scale-2, Beck Depression Inventory II, and the Columbia Suicide Severity Rating ScaleThe following assessments will be completed to screen for study/surgical eligibility:MDS-UPDRSFOG QuestionnaireTimed Up and Go (TUG) testStride length Velocity Gait variability Instrumented gait assessment  measuring:360° clockwise and counterclockwise turn on the spot (time and number of steps)The Parkinson’s Disease Questionnaire (PDQ-39) and the Parkinson’s Disease Quality of Life Questionnaire (PDQL)Columbia Suicide Severity Rating ScaleA brief pain history and Neuropathic Pain Symptom InventoryBlood pressure and postural drop assessed in the off and on DBS stimulation state.The following assessments will be completed prior to DBS implantation as well as at each clinic visit post-implantation:At study conclusion, patients will undergo a detailed kinematics gait assessment using the Nexus system (Vicon Motion Systems Ltd) and surface EMG recordings in the lower limbs.The Vercise™ (Boston Scientific Corporation) DBS System and Cartesia™ (Boston Scientific Corporation) directional leads will be used in this study. Implantation of electrodes and generator will be performed in one procedure, as previously described at our institution  but targIntraoperative stimulation parameters will be used to guide initial programming. Programming sessions will have heart rate, blood pressure, respiratory rate, and blood oxygenation saturation monitoring to mitigate the risk of adverse cardiorespiratory events related to stimulation. Additionally, low frequencies will initially be explored with patients until an adequate stimulation amplitude is found where gait initiation is under volitional control. Stimulation amplitude will then be increased until an adequate gait speed is achieved. If this is not immediately apparent, stimulation amplitude will be increased to just below the threshold for onset of side effects. Based on other studies, cyclic stimulation with continuous daily stimulation and night arrests will be the default protocol to avoid habituation and waning of effects , 54. CurThe analyses for safety will be descriptive, focusing on trends for within-subject differences and changes in FOG symptoms or off-target side-effects during the duration of the study. All adverse events (AEs) will be listed and their incidence compared to historical controls. The primary safety analysis will be conducted on all patient data when all 4 participants have completed the study.DeathOccurrence of pre-defined stoppage rulesSuccessful implantation and complete follow-up of 4 patientsOutcomes of neurological and functional status, gait, and quality-of-life are collected at several time points during the study Table .Percent change in FOG Questionnaire (FOGQ), Parkinson’s Disease Questionnaire (PDQ-39), and the Parkinson’s Disease Quality of Life Questionnaire (PDQL)Percent change in Movement Disorders Society Unified Parkinson’s Disease Rating Scale (MDS-UPDRS) Part III across study visits relative to pre-operative baseline evaluation.Percent change in number of falls Percent change in gait velocity with and without CnF DBSPercent change in muscle electromyogram (EMG) amplitude during gait testing at study conclusion between CnF DBS on and off conditionsn = 4). Subjects will be fitted with fifteen reflective markers that will be tracked at 100 Hz with a 10-camera Nexus system (Vicon Motion Systems Ltd) attached bilaterally to the first and fifth distal phalange of the foot, lateral malleolus, calcaneus, lateral femoral epicondyle, anterior superior iliac spine, and the mid-shank and mid-thigh. A reflective marker will also be placed on the lower back region overlying the sacrum. EMG surface electrodes will be placed over the tibialis anterior (TA) as well as the lateral gastrocnemius (LG). Subjects will be asked to walk along a 25-foot path at a constant, regular walking pace both without and then with optimal DBS configuration settings (CnF L on/CnF R on), DBS OFF (CnF L off/CnF R off), DBS RIGHT Only (CnF L off/CnF R on), and DBS LEFT only (CnF L on/CnF R off) repeating each condition twice. Kinematic and EMG signals will be recorded during each trial while the subjects walk for 25 ft. and averaged for each test condition.Lower-limb gait kinematics and EMG will be evaluated with and without DBS in study subjects  at each clinic visit, with the Tukey Honest Significant Difference post hoc test to compare configurations. The same test will be used to compare changes in EMG amplitude between the four DBS configurations at the final gait assessment at the study’s conclusion. One-way ANOVA with repeated measures will be used to compare changes in the UPDRS III, FOG Questionnaire, PDQ-39, and PDQL over the multiple time points of the study.All study documents and files will use an anonymous study identification number to identify subjects and only an appointed Study Coordinator will maintain linkages, to maintain subject confidentiality. Additionally, locally stored computer data will be password protected and located within our institution-based firewall. Physical study data files including signed consent forms will be stored securely in the principal investigator’s (JJ) office within a locked filing cabinet. Subject data will be documented in source documents initially and then recorded on electronic case report forms (CRFs). The Study Coordinator will ensure that data is entered into CRFs within 5 days of data collection and will review the accuracy of entered data by comparison with subjects’ medical records within 30 days. Potential discrepancies will be flagged and checked and corrected as necessary by study investigators.Only personnel listed on study protocols will have access to study data, though study data that could medically benefit subjects will be shared with them. There will be periodic audits (at least two during the study) of the data by university’s Office of Research Compliance and Quality Assurance (RCQA) auditors to ensure compliance with the FDA’s Good Clinical Practices and ICH-E6. The Principal Investigator will submit all de-identified CRFs to the Study Sponsors and the FDA throughout and at the completion of the study, including any incomplete CRFs and CRFs of those who withdraw before study completion.A Data Safety and Monitoring Board (DSMB) has been established as an independent expert advisory group to assess adverse events (AEs) and evaluate the general integrity and conduct of the study. The DSMB chair will be provided a monthly update on the status of all study subjects. Furthermore, all AEs related to the study will be reported to the DSMB, which will make recommendations as to whether the study should continue without change, be modified, or be terminated early. Pre-determined stoppage rules have also been established to aid with early termination decision making Table . No formPD patients with FOG that is refractory to dopaminergic therapy have limited therapeutic options. Our study protocol aims to test the safety, feasibility, and preliminary efficacy of a novel brainstem target to alleviate FOG. Our strategy is based on a careful revisiting of classical animal studies , newly eA strength of this study is the novel use of directional electrodes to deliver DBS to this midbrain region. As a complex area comprised of numerous interleaving fiber tracts and structures, the ability to steer current would be important in optimizing therapeutic effects while mitigating stimulation-induced side effects. Limitations of this study include the small sample size, given the preliminary nature of this study, and the lack of a control cohort. Additionally, as some patients with DBS in this region feel mild paresthesias with stimulation, complete double blinding of DBS status during gait assessments may be impossible. Finally, there are limitations related to the studying of FOG itself, including potential heterogeneity of pathobiology, and difficulty in eliciting FOG in the clinical setting. We hope to mitigate these difficulties through our rigorous inclusion/exclusion criteria, and by using known triggers for FOG during gait assessments, if necessary.Study protocol version 2 was approved March 2, 2020, by the FDA. Recruitment started April 27, 2020, but was delayed due to the COVID-19 pandemic and is expected to be completed by December 31, 2022.


Persistent low back pain (LBP) is a major health issue, and its treatment remains challenging due to a lack of pathophysiological understanding. A better understanding of LBP pathophysiology has been recognized as a research priority, however research on contributing mechanisms to LBP is often limited by siloed research within different disciplines. Novel cross-disciplinary approaches are necessary to fill important knowledge gaps in LBP research. This becomes particularly apparent when considering new theories about a potential role of changes in movement behavior (motor control) in the development and persistence of LBP. First evidence points toward the existence of different motor control strategy phenotypes, which are suggested to have pain-provoking effects in some individuals driven by interactions between neuroplastic, psychological and biomechanical factors. Yet, these phenotypes and their role in LBP need further validation, which can be systematically tested using an appropriate cross-disciplinary approach. Therefore, we propose a novel approach, connecting methods from neuroscience and biomechanics research including state-of-the-art optical motion capture, musculoskeletal modeling, functional magnetic resonance imaging and assessments of psychological factors. Ultimately, this cross-disciplinary approach might lead to the identification of different motor control strategy phenotypes with the potential to translate into clinical research for better treatment options. Low back pain (LBP) is one of the most common conditions regarding years living with a disability throughout the world . The preSo far, research on LBP has identified several pathogenic mechanisms involving biophysical, genetic, social and psychological contributors . ResearcHowever, as recently stated, research on these different pathomechanisms of LBP is often limited by significant knowledge gaps arising from siloed research within different research disciplines, highlighting the need for cross-disciplinary approaches that have the potential to identify important interactions between different mechanisms contributing to LBP . This beTherefore, after a summary of the relevant literature, we present a novel cross-disciplinary approach combining neuroscientific and movement biomechanics research methods with the aim of identifying different motor control strategy phenotypes and their role in LBP as well as their underlying supraspinal, psychological, and biomechanical features. Ultimately, this approach might help to fill important knowledge gaps in LBP research with the potential to translate into clinical research for better treatment options.Numerous studies have investigated biomechanical alterations in LBP, mainly by observing spine/trunk kinematics and muscle activity during functional activities as well as during steadily held postures with and without experimentally induced perturbations. Investigations of functional activities in LBP patients compared to healthy controls indicate trends toward a reduced lordotic posture and range of motion (RoM) in the lumbar spine during activities such as standing, walking, running, chair rising or picking up an object –20. In tMore than 20 years ago and using magnetencephalography, researchers detected a shifted sensory representation of tactile input from the back in chronic LBP patients in the primary somatosensory cortex . MoreovePain-related fear and associated avoidance behavior as well as depression and anxiety have received extraordinary attention in the last two decades because they were empirically identified as important psychological factors in the development and persistence of LBP , 39, 40.To investigate potential interactions between psychological factors, biomechanical mechanisms and supraspinal processes in LBP , we propTo assess pain-related fear, self-reports are an adequate direct measure of subjective feelings of fear that are easily accessible for clinicians and researchers . The mosThe functional biomechanics of the spine are investigated using a comprehensive non-invasive experimental and computational approach, which combines state-of-the-art optical motion capture with advanced musculoskeletal modeling. Motion data are collected in a motion analysis laboratory, where participants are equipped with 58 retro-reflective skin markers according to a previously developed configuration  Figure  and askein vivo measurements  measurements of the main trunk stabilizers and to use this information as additional input for our models. This will further increase prediction accuracy, especially when participants present activity patterns such as increased antagonistic muscle coactivation, which was shown to have direct implications on spinal loading , 23.Non-invasive human brain imaging techniques such fMRI with its high spatial resolution provide suitable tools for the investigation of the cortical representation of different body parts . We deveUsing the pneuVID in combination with high spatial resolution fMRI (3 or 7 Tesla), detailed cortical maps of paraspinal afferent input can be explored using different vibration frequencies: Applying vibratory stimulation at frequencies between 60 and 80 Hz and amplitudes of 0.5–1 mm on paraspinal muscles has been shown to be a potent stimulus for muscle spindle activation  . In contUsing the methodologies spanning different research disciplines as described above, the current approach has the potential to address important questions in LBP research:Do loose/tight motor control strategy phenotypes indeed exist and/or do other motor control strategies exist? Biomechanical assessments of dynamic movement tasks, involving subject-specific spine kinematics, segmental loadings and paraspinal muscle forces during daily activities , will be performed to investigate potential relationships with LBP duration, disability, and psychological factors. Relevant features will be extracted for subsequent data analysis  with the goal of classifying different motor control strategy phenotypes that are possibly associated with different LBP symptom durations .via neuroplastic cortical changes   is causally linked to LBP-provoking alterations in motor control t input) . For theAs recently stated, a better understanding of musculoskeletal pain depends on reconnecting the brain with the rest of the body . Our appProvided that the suggested motor control strategy phenotypes can be reliably identified using the approach described in this article, the knowledge generated might lead to important implications for clinical research and interventions. For example, it has been proposed that a persistent “tight control strategy” may be specifically targeted by reducing muscle excitability and co-contraction while increasing movement variability in motor control exercise . With reThe original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.SS and MM wrote the first draft of the manuscript. CB and PS critically revised the manuscript and contributed additional text parts. All the authors approved the version to be published.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The practice of medicine has steadily employed less invasive methods to obtain information derived from the tumor to guide clinical management of patients. Liquid biopsy—the sampling of blood—is a non-invasive method for generating information previously only available from tissue biopsies of the tumor mass. Analysis of fragmented circulating tumor DNA in the plasma is clinically used to identify actionable mutations and detect residual or recurrent disease. Plasma analysis cannot, however, assess cancer phenotypes, including the expression of drug targets and protein biomarkers. Circulating tumor cells (CTCs) are intact cancer cells that have entered the blood that have the potential for distant metastasis. While enumeration of CTCs is prognostic of outcome, recently developed technology allows for the interrogation of protein biomarkers on CTCs that could be predictive of response. Furthermore, since CTCs contain intact whole cancer genomes, isolating viable CTCs detected during therapy could provide a rational approach to assessing mutational profiles of resistance. Identification, characterization and molecular analysis of CTCs together will advance the capacity of liquid biopsy to meet the requirements of twenty-first century medicine. Pathology is the discipline of medicine concerned with the analysis of tissues and body fluids to diagnose disease and so guide its treatment. Within oncologic pathology, there has been a steady historical trend in tumor tissue sampling and examination. Advances in methods and technologies have allowed access to tissues by diagnostic approaches that are increasingly less invasive to the patient: from tumor resection to excisional biopsy, incisional biopsy, core needle biopsy and then to fine needle aspiration biopsy. In the twenty-first century, precision treatments are directing therapy to the molecular drivers and differentiators of cancer rather than treatment based on the tissue of origin and morphologic subtype. Thus, increased amounts of molecular information at the protein expression  and nucleic acid sequence (actionable mutations) level are demanded from biopsy samples even as the volume of the available material decreases.EGFR and PIK3CA to guide targeted therapies, and gene expression panel tests for determination whether chemotherapy should be included in the treatment of hormone receptor-positive breast cancer.After morphologic diagnosis, the biopsy sample may be used to identify phenotypic molecular sub-classifications that inform prognosis, to generate biomarkers of therapeutic response, and for companion diagnostic testing. Examples include estrogen, progesterone, and HER2 receptor testing in breast cancer to define hormonal or HER2 driven cancers; the use of the proliferation marker Ki-67 to characterize aggressiveness of cancer growth; and the testing of levels of PD-L1 expression that support treatment with immune checkpoint inhibitors. In addition, the biopsy sample is used for nucleic acid analysis at both the DNA and RNA level. Examples include sequencing of DNA for identification of mutations in receptors such as The least invasive way to access a tumor is by sampling blood—a “liquid biopsy”—rather than the tumor itself. In clinical practice this typically refers to circulating tumor DNA (ctDNA) based testing of plasma samples. However, the term broadly includes other elements of cancer that can be detected in the blood, including circulating tumor cells (CTCs). There are many advantages of liquid biopsy testing, including: 1) ease of access to samples; 2) minimal procedural discomfort and complications (considered non-invasive); 3) integration of tumor information from various locations throughout the body; 4) real-time data rather than historical snapshot; and 5) longitudinal sampling. Serial sampling allows for better assessment of intra-tumoral as well as temporal-heterogeneity in cancers, especially ones exposed to targeted therapies rather than chemotherapy . Analysieach distant tumor metastasis was once a CTC. To spread to a distant site  a cancer cell must enter the blood stream. Since it is estimated that more than 90% of cancer mortality is caused by distant metastasis, understanding and counteracting this process will be essential to managing cancer. Investigation of CTCs is likely to provide relevant information, since these cells are already “halfway” to metastasis: they have broken free from the tumor mass, transited through interstitial tissues, and intravasated into the vasculature. The second point is that viable CTCs that are present during cancer therapy are resistant to treatment. CTCs therefore represent a population of cells that can be investigated for mechanisms of resistance even if the therapy is successful in reducing tumor burden. These mechanisms may be phenotypic as well as genotypic. CTC liquid biopsy thus collects tumor cells with both metastatic and resistance potential. The incidence and number of detected CTCs is associated with cancer type and stage, increasing with more advanced disease. In metastatic cancers, the incidence of CTC-positive patients can be greater than 50%. Consequently, the use of CTC analysis in clinical research and practice is most appropriate for later stage disease  which can be important for understanding cell physiology. Finally, a method for isolation of individual cells is necessary. RareCyte has developed a platform and workflow that combines these features to support the next generation of cell-based liquid biopsy applications .® system—blood collection, nucleated cell density separation, and slide preparation); immunofluorescence [IF] staining (RarePlex® kits); and imaging . Briefly, the buffy coat fraction of the blood containing CTCs is separated and spread in a monolayer smear onto microscope slides; the slides are stained by IF using automated stainers; the stained slides are scanned, and image files are analyzed by automated software to define candidate CTCs for confirmation by a trained reviewer. Identification of epithelial CTCs requires three IF channels—1) cocktail of anti-pan-cytokeratin (CK) and anti-EpCAM antibodies; 2) anti-CD45 antibody; 3) nuclear dye. Two additional channels 4) and 5) are used for biomarkers that are included in validated assays or can be used for investigation novel markers using a kit that allows insertion of antibodies. A CTC must show staining in the CK/EpCAM channel, no staining in the CD45 channel, and must have a nucleus. Machine learning systems assist the reviewer by rank ordering candidate cells based on probability of being a CTC. CTC nucleic acid sequencing is accomplished in an additional final step in which identified CTCs are mechanically removed from the slide  and placed individually into tubes.The CTC analysis workflow includes four steps. The first three allow CTC enumeration and protein biomarker analysis: sample processing . In EMT cells lose epithelial markers and take on mesenchymal ones, and it is increasingly understood that there are hybrid cells that express both phenotypes simultaneously ; such E/Additional biomarker channels enable investigation of markers that may be known to be clinically significant within a tumor type, or of investigational interest. For instance, we have qualified and analytically validated CTC assays for androgen receptor (AR), AR splice variant 7, and synaptophysin (prostate cancer), HER2 and ER (breast cancer), and PD-L1 (immune-oncology). In addition, we have qualified additional markers, including PSMA, PSA, Muc1, progesterone receptor, EGFR, HER3 and Ki-67 for various epithelial cancers. These markers can be used in combination to define phenotypic subsets of cells. For instance, breast cancer CTCs can be divided into ER+/HER2+, ER+/HER2-, ER-/HER2+ and ER-/HER2-groups and their presence followed over time. Not all protein biomarker analyses would be of interest to all tumor types. The flexibility of the platform allows clinicians and investigators to choose the relevant markers of choice. For example, in patients with gastrointestinal (GI) malignancies, the hormone receptors have no role. However, other markers are of high interest, including EGFR and HER2, which has had intense drug development surrounding it over the past several years. The ability to assess this marker serially by blood sampling could prove highly useful, since tumors can gain or lose HER2 over time .There are clinical situations in which patients cannot undergo invasive diagnostic procedures because of risk, infrequent healthcare visits, cost or lack of access to advanced technology. In these settings, routine tissue biopsy procurement may be impossible. For example, some patients with a suspicious lung imaging lesion consistent with cancer cannot undergo bronchoscopy due to procedural risk; other patients with clinical symptoms consistent with cancer (such as long-term smoking exposure) may not have access to standard imaging for diagnostic evaluation; in global health settings invasive surgical procedures may not available. The detection of CTCs via a blood sample that includes confirmatory markers of tissue origin  may establish a sound medical rationale for confirmation of a cancer diagnosis and subsequent treatment.Drug targets and protein markers of therapeutic efficacy can be evaluated on actual tumor cells in the blood using CTC-based liquid biopsy. This provides a rational approach to the development of non-invasive companion diagnostics that are not sequence based.Lineage differentiation biomarkers can be employed to ascertain the transformation of a tumor from one sub-type to another. For example, a significant fraction of castration resistant prostate cancers will transform to neuroendocrine (NE) prostate cancer which no longer responds to anti-androgen therapies. NE differentiation can be detected by expression of synaptophysin on CTCs, offering a rational minimally invasive alternative to procurement of tissue  for confirmation of transformation. Another type of transformation is epithelial-to-mesenchymal transition, which is understood to be an important aspect of cancer progression and metastasis. Current consensus is that cells are rarely purely mesenchymal, but typically retain some epithelial characteristics while gaining mesenchymal ones. By adding markers such as vimentin or N-cadherin to the epithelial CTC assay, transformation to mesenchymal phenotype can be followed.Understanding drug-target interaction at a molecular level via sequential tumor biopsies is exceedingly difficult in clinical studies. Yet it is extremely important to understand whether an investigational drug is having meaningful effect on the cells it is intended to treat. CTC biomarker assays make possible the assessment of drug activity via downstream markers known to be modulated by drug action.Most studies of CTCs are in populations of patients with epithelial tumors, that together constitute most cancers. First generation CTC technologies have used antibodies to epithelial surface markers  to capture CTCs from the blood, which can then be confirmed by visualization of a second common epithelial marker, such as cytokeratin. This has been effective in identification of CTCs that express both markers, although CTCs that express only one will be missed using this approach. Not all cancers are epithelial, however (in fact the first reported case of circulating tumor cells by Ashworth in 1869 was in a patient with a sarcoma), and CTCs from non-epithelial cancers will not be detected using epithelial markers. This limitation can be overcome with a platform that allows flexible substitution of other markers for specific identification of the cell type. For example, RareCyte has qualified antibodies to S-100, melan-A, and NG2 for identification of circulating melanoma cells.Cell-free DNA is most effectively employed for targeted sequencing of circulating tumor DNA (ctDNA). However, ctDNA analysis is not always successful; allelic fraction and total amount of ctDNA must be sufficient for confident mutation detection. CTCs contain “pure” cancer genomes, minimizing if not eliminating the influence of allelic fraction on sensitivity. The RareCyte platform has been used to identify actionable PIK3CA mutations in CTCs that were not identified in plasma using the same targeted panel in patients with metastatic breast cancer .CTCs contain the entire intact cancer genome, allowing genome-wide sequencing that can be employed for broad detection of mutations. The RareCyte platform has been used together with whole exome sequencing to follow the genomic evolution of CTCs in triple-negative breast cancer over the course of 9 months . Since mApoptotic cells are understood to release ctDNA that is detectable in the plasma in short fragments that can be tested for mutations. Resistant clones may comprise only a small fraction of the entire tumor mass, and they are less likely to undergo apoptosis, so their genomes may not be detected at the current limit of sensitivity of cell-free DNA assays. As discussed above, there is a rational basis for higher representation of resistant clones within the viable CTC population. When patients are undergoing therapy, CTCs present may be identified, isolated, and sequenced to find actionable mutations within the resistant population are not present in the bulk of the tumor to guide planning for subsequent treatment.ctDNA analysis can assess mutational heterogeneity across a bulk population of tumor cells but cannot resolve mutational profiles within individual cells. Heterogeneity is inferred by presence of different variant allele fractions of mutations, which often tend to be subclonal . Since CIn a clinical study of patients with advanced gastrointestinal cancers, CTCs were counted and followed during treatment. CTCs from a colorectal cancer patient were retrieved from slides after imaging for hotspot sequencing. The presence of an APC nonsense mutation was detected in three of eight CTCs. This is described in detail in The promise of liquid biopsy is that it will provide the types of valuable information for research and clinical care that currently require tissue biopsies. The many advantages of cell-based liquid biopsy have been discussed in the introduction, but there are limitations. Not all cancer patients have CTCs in the volume of blood typically used for analysis (<10 ml), especially in early clinical stage disease. Yet for many patients, especially those with advanced or recurrent disease, CTCs are not limiting, and thus are a source of cancer tissue for serial real-time analysis. It is important to use highly sensitive detection technologies that are integrated with analytic methods for protein and nucleic acid analysis.Evaluation of cellular protein biomarkers in tissues is part of standard pathology laboratory practice. Many clinically important protein biomarkers cannot be assessed by sequence, and so require cancer cells for assessment rather than soluble biomolecules. Technologies and methods have been developed that robustly identify CTCs and can assay multiple biomarkers on them. This has the potential for broad impact in tumor characterization and diagnostic confirmation, companion diagnostics development, monitoring of tumor transformation, pharmacodynamic investigation, non-epithelial cancers, and minimal residual disease monitoring.Sequence and gene expression analysis of tissue samples to identify cancer mutations and profiles is routinely performed by molecular pathology laboratories. The fact that CTCs have a complete cancer genome and transcriptome, are critical to cancer metastasis, and may be enriched for resistant clones, suggests that genomic and transcriptomic analysis of CTCs may provide useful information that is not available in ctDNA. Since plasma and CTCs can both be isolated from the same blood collection tube, a strategy of plasma sequencing reflexing to CTC sequencing in cases where the ctDNA result is non-informative is a rational approach to increasing diagnostic sensitivity. This workflow is consistent with the concept that liquid biopsy methods should not be considered mutually exclusive. Similar to how liquid biopsies have not replaced tissue biopsies, CTC testing is not intended to replace ctDNA-based testing. Rather, the different approaches are best understood as complementing one another to provide a more complete assessment of cancer.We have described a variety of CTC protein biomarker and sequencing applications in research and diagnostics based on an established liquid biopsy technology platform. There are other specimen types that can be classified as “liquid biopsy” that are routinely processed in the cytology laboratory. These include body fluids  and fine needle aspirate biopsies in which cellular fluid is aspirated from a tumor. Slide smears or monolayer preparations are made for morphologic evaluation and molecular testing. These small-volume samples can be difficult to thoroughly evaluate by traditional methods that require multiple slides for immunohistochemistry or sufficient material for sequencing. But since they are similar to buffy coat smears, they could be analyzed using the liquid biopsy methods described here for multi-protein biomarkers and target cell sequencing .The promise of liquid biopsy is that it will be able to do what tissue biopsy has done—generate essential information for the diagnosis, phenotyping and molecular analysis of cancer. The challenge for liquid biopsy is to combine appropriate technologies with robust assay methods that can extract that information from blood components. ctDNA sequencing has rapidly gained adoption as a mutational testing method. We have described here how the phenotypic and molecular analysis of CTCs can supplement ctDNA sequencing to advance the capacity of liquid biopsy to meet the requirements of twenty-first century medicine.


BPaL, a 6 month oral regimen composed of bedaquiline, pretomanid, and linezolid for treating extensively drug-resistant tuberculosis (XDR-TB) is a potential alternative for at least 20 months of individualized treatment regimens (ITR). The ITR has low tolerability, treatment adherence, and success rates, and hence to limit patient burden, loss to follow-up and the emergence of resistance it is essential to implement new DR-TB regimens. The objective of this study was to assess the acceptability, feasibility, and likelihood of implementing BPaL in Indonesia, Kyrgyzstan, and Nigeria.We conducted a concurrent mixed-methods study among a cross-section of health care workers, programmatic and laboratory stakeholders between May 2018 and May 2019. We conducted semi-structured interviews and focus group discussions to assess perceptions on acceptability and feasibility of implementing BPaL. We determined the proportions of a recoded 3-point Likert scale , as well as the overall likelihood of implementing BPaL  that participants graded per regimen, pre-defined aspect and country. We analysed the qualitative results using a deductive framework analysis.In total 188 stakeholders participated in this study: 63 from Kyrgyzstan, 51 from Indonesia, and 74 from Nigeria The majority were health care workers (110). Overall, 88% (146/166) of the stakeholders would likely implement BPaL once available. Overall acceptability for BPaL was high, especially patient friendliness was often rated as acceptable . In contrast, patient friendliness of the ITR was rated as acceptable by 45%. Stakeholders appreciated that BPaL would reduce workload and financial burden on the health care system. However, several stakeholders expressed concerns regarding BPaL safety (monitoring), long-term efficacy, and national regulatory requirements regarding introduction of the regimen. Stakeholders stressed the importance of addressing current health systems constraints as well, especially in treatment and safety monitoring systems.Acceptability and feasibility of the BPaL regimen is high among TB stakeholders in Indonesia, Kyrgyzstan, and Nigeria. The majority is willing to start using BPaL as the standard of care for eligible patients despite country-specific health system constraints.The online version contains supplementary material available at 10.1186/s12889-021-11427-y. Despite increasing access to new diagnostics and anti-tuberculosis (TB) medicines, treatment success rates for extensively drug-resistant TB (XDR-TB) remain low and contribute to the ongoing transmission of DR-TB . GloballThe Global Alliance for TB Drug Development (TB Alliance) has developed a 6 month oral regimen consisting of three drugs: bedaquiline (Bdq), the new drug pretomanid, and linezolid (Lzd). This BPaL regimen was successful in over 90% of patients with XDR-TB, multidrug resistant TB (MDR-TB) treatment failure, or intolerance to MDR-TB treatment in the Nix-TB trial . Side efImplementation of the BPaL regimen would entail essential changes in clinical and programmatic TB management . The redTo ensure rapid adoption after the regimen is endorsed by regulatory authorities, evidence is needed on health care system readiness, barriers and requirements among those implementing the intervention –13. The We used a concurrent mixed methods design. The qualitative component, consisting of semi-structured interviews and focus group discussions (FGD) among stakeholders, explored the acceptability and feasibility of seven aspects of PMDT, comparing ITR and BPaL. The seven aspects of PMDT were: patient baseline assessment and monitoring of treatment efficacy, treatment safety monitoring, patient friendliness, patient support, programmatic aspects, human resources, and procurement and supply chain management (PSCM).For the quantitative component of the study, we used a Likert scale to quantify how acceptable stakeholders perceived each PMDT aspect for ITR and BPaL, as well as the overall likelihood of implementing BPaL. We triangulated the two data types to explore if quantitative findings could be explained by the qualitative results.We conducted the study from May 2018 – May 2019 among health care workers , programmatic stakeholders  and laboratory stakeholders  in Indonesia, Kyrgyzstan, and Nigeria. Target participant numbers per subgroup are shown in Supplementary file We chose Indonesia, Kyrgyzstan, and Nigeria to depict various geographical settings , with differences in TB burden  and DR-TB) and health systems infrastructure. The estimated TB incidence per 100,000 population in 2018 was 316 in Indonesia, 116 in Kyrgyzstan and 219 in Nigeria . The estThe conceptual framework from Atun et al.  guided tWe developed the interview guide and questionnaire  and PMDT aspect. We also determined the proportion of stakeholder that chose likely, neutral and not likely per scenario of implementing BPaL as SoC. We stratified results by country.A total of 194 stakeholders participated in this study. Four Kyrgyz participants were excluded from all analyses due to inaudible recordings and two Kyrgyz participants were excluded due to declined consent for recording. Three stakeholders in Indonesia and three in Nigeria did not fill out the questionnaire for the quantitative part. Thus in total, 188 stakeholders were included in the qualitative data analysis and 182 in the quantitative analyses (97 and 94% respectively)  as the Nigerian health care worker: “ … (compared to the) individualized treatment regimen (there are many) benefits are … no matter how somebody tries, taking a drug for 20 months is not easy … in terms of adherence, … out of pocket cost, … side effects … (with the 6 months regimen) they (the patients) are going to be treated quickly and … resume their normal economic activities … I think there are more benefits for them.” [FGD]Another benefit was that stakeholders believed that the BPaL regimen would increase patients’ quality of life during treatment and would improve treatment adherence, which would contribute to higher treatment success (Table Indonesian caregiver: “Side effects (of BPaL are) … much less … compared to the individualized (regimen) … so for the patient’s level of … adherence … perhaps it’d be better because what happens so far, the patient cannot bear the side effects and becomes lost to follow up... he stops taking the medication.” [interview]Besides the reduction of side effects being a benefit, a challenge participants opted was about the side effects of (high-dose) Lzd, including myelosuppression (bone marrow suppression (which leads to reduced production of blood cells) and peripheral neuropathy  , it is very low the patient is dying slowly...” [FGD]In terms of benefits, stakeholders perceived BPaL to be more patient-friendly compared to the ITR resulting from its shorter treatment duration, lower pill burden per day, fewer anticipated side effects, lower financial burden, decreased requirements for higher level of care, and reduced patient discomfort (including risk of hearing loss) due to the removal of injectables Table .Nigerianss Table .Indonesin) Table . StakehoIndonesian health care worker: ( … ) we should pay attention to ( … ) supervise them (the patients) taking ( … ) the drugs … We have to provide counselling for the patients to remind them the importance of taking the drugs. It is the same.” [FGD]Participants thought the BPaL regimen would allow cutting costs for patient support due to its shorter duration, while they thought the type/form of patient support required was the same Table , includiIndonesian Programmatic Stakeholder: “The initial phase and this would be a challenge if it [BPaL] is (implemented) in Puskesmas [Indonesian primary health centres], in an outpatient setting … because we must still separate the patients that have been (tested) negative from the patients who are still positive..” [Interview]Despite the anticipated cost reduction for DR-TB treatment with BPaL, stakeholders from all countries considered current high costs of Bdq and Lzd a challenge . Some Kyrgyz and Nigerian stakeholders had experienced supply challenges with the introduction of the shorter regimen , Bdq and Delamanid (due to lack of registration). They therefore had concerns about sufficient supply of new drugs.Kyrgyz health care worker: “So, its (safety) is unclear yet. For example, with linezolid we notice that side effects are irreversible and with pretomanid we don’t know are they reversible or irreversible... it is not studied yet … ” [Interview]Indonesian and Nigerian stakeholders expressed challenges about current health systems gaps, especially at lower health care levels where BPaL may be implemented. These challenges included limited availability of ancillary drugs (Indonesia) and currently weak monitoring systems for regular haematology and electrocardiogram (ECG) (Nigeria). Stakeholders opted as practical requirement that safety monitoring and side effect management should be guaranteed and provision of ancillary drugs should be free of charge:Nigerian programmatic stakeholder: “We will have to strengthen our system to ensure that patients’ baseline and follow up investigations are done … when they are due so that we will be able to pick up any side effect at an early stage. If that is done, it will make it (BPaL) more friendly, but when that is not done patients will run away from it.” [Interview]For monitoring of treatment safety, stakeholders considered it a benefit that BPaL has shorter duration and less audiometry requirements. However, many also perceived challenges due to uncertainties about safety, possible need for intensified monitoring due to the high-dose of Lzd in the regimen and lack of experience with pretomanid:Nigerian health care worker: “In case we adopt the BPaL and somebody has a serious adverse event (so) that you need to discontinue the linezolid, then what will happen to the potency of the regimen? … If you need to discontinue one of the drugs, what happens to the efficacy of the regimen?” [FGD]Another major challenge expressed by Kyrgyz and Nigerian stakeholders was on safety concerns and the applicability of BPaL among children, pregnant women, and patients with comorbidities.Although some participants perceived the current expected low prevalence of drug resistance to drugs in the BPaL regimen as beneficial, participants from all countries had considered the emergence of resistance to BPaL or its components as a challenge. Nigerian health care workers were also concerned about baseline Lzd resistance as the drug is widely used for other diseases. Other challenges mentioned were about dose related Lzd intolerance and efficacy of the regimen when continuing treatment with only two drugs contributing to resistance to the remaining drugs or weakened regimen efficacy:In Indonesia and Nigeria, the lack of diagnostics and Drug Susceptibility Testing (DST) capacity for regimen allocation was expressed to be an important health systems challenge. This included the lack of access to GeneXpert MTB/RIF and/or second-line line probe assay testing in some areas, and the lack of DST capacity for Bdq and Lzd with liquid culture or genetic sequencing on a large scale.Kyrgyz programmatic stakeholder: “Every patient should have an individual approach. Seriously ill patients with side effects, with additional comorbidities they need a completely different approach.” [Interview]Moreover, stakeholders stressed the importance of introducing BPaL under operational research conditions. The need was expressed for clear local guidelines, capacity building, and training on the use of BPaL. Stakeholders flagged the need for training on monitoring and management of DR TB treatment, related adverse events, and guidance on dealing with patients who fail on a regimen. Clear guidelines on Treatment efficacy- and safety monitoring, management of adverse events, as well as patient support would be needed to support decentralized implementation.Stakeholders expressed the need for clinical studies in certain groups and in a national context. They expressed the need for adjusted treatment guidelines among specific groups:Other crucial practical requirements expressed for BPaL implementation were the needs for diagnostic capacity for DR-TB, regimen design including the drug composition of only three drugs Table . PSCM plWe assessed the acceptability and feasibility of implementing BPaL among key stakeholders in TB care and management from Indonesia, Kyrgyzstan, and Nigeria. Study participants found BPaL more acceptable than the ITR because of anticipated benefits such as improved patient-friendliness and reduced health system burden as a result of increased patient-friendliness, likelihood of improved adherence and anticipated reduced cost of BPaL. Additionally, stakeholders considered the implementation of BPaL feasible within the local health care infrastructure if efforts are undertaken to address practical requirements such as gaps in current treatment and safety monitoring systems that have become more apparent with introduction of recent new drugs and regimens as well as efficient diagnostic and DST capacity.Triangulating the qualitative and quantitative results indicated higher overall patient-friendliness score for BPaL (93%) in comparison to the ITR (45%) could be linked to stakeholders’ anticipated patient challenges with the current ITR which will be improved with BPaL, i.e. shorter duration, absence of injectables, lower pill burden, and lower financial burden to the patient. The shortened treatment duration and reduced costs may explain the higher overall acceptability of BPaL patient support in comparison to the ITR (85% vs. 69%). The higher overall acceptability of programmatic aspects of BPaL (83%) as compared to the ITR (60%) might be linked to the anticipated benefits of improved treatment outcomes as well as reduced financial burden to the health system due to the shorter treatment duration and increased possibility for the decentralization of treatment provision. Higher acceptability scores for the baseline assessment and treatment efficacy monitoring may be driven by stakeholders perceiving BPaL as beneficial in terms of being less burdensome and cost-saving as compared to the ITR. The higher acceptability for human resources for BPaL (79% vs. 59%) could be linked to the benefit of reduced burden on health care workers. The higher acceptability of PSCM (BPaL 80% vs. ITR 67%) may be explained by stakeholders’ positive expectation that PSCM will become simpler, with only three drugs compared to the 15 in ITR. Acceptability ratings in Indonesia may be lower due to concerns about complexities relating to local regulatory requirements and approvals, along with requirements to procure linezolid locally at a very high price. Similar acceptability scores on the treatment safety monitoring on BPaL and ITR may be explained by the expected benefits such as shorter duration (resulting in less treatment monitoring visits and actions) being offset by challenges relating to BPaL safety, and current health systems gaps.Our findings aligned with those of Thomas et al. , and VegMany stakeholders were especially concerned about the side effects of Lzd, including myelosuppression and neuropathy. The ZeNix trial is assessing the efficacy, safety and tolerability of various doses and durations of Lzd in the BPaL regimen . ConsequThe stakeholders showed concern regarding the potential emergence of resistance to novel drugs such as Bdq, which highlights the need for development of rapid DST for new medicines . CurrentThis study has some limitations. We did not include patients in this study. We aimed to represent their experiences indirectly by interviewing health care workers. As per the design of the assessment, many questions regarding the BPaL regimen were of a hypothetical nature and participants did not have any practical experience with BPaL at the time they were interviewed. Therefore, there may have been misconceptions leading to biased perceptions around BPaL. To mitigate these biases we provided each participant with standardized information about BPaL based on the available evidence from the Nix-TB trial. We used convenience sampling for selection of our interviewees. We acknowledge that this method may lead to a non-random sample. We aimed to limit this bias by inviting participants based on a clear guidance on expertise and positions. Another limitation was that the data collection tools were only piloted in Nigeria, the translated tools were not piloted which could have caused misinterpretation due to lingual and cultural differences. Each country employed at least 1 but no more than 3 interviewers to conduct interviews. This may have caused variability, which we tried to minimize by training of interviewers and standardizing semi-structured questionnaires. We addressed this by having an interviewer present who speaks the local language and as we reached data saturation, we do not expect the variability to have limited the quality and richness of our qualitative data.To our knowledge, this is the first study to assess the acceptability and feasibility of the implementation of the BPaL regimen compared to current treatment options, thus providing unique insights from a health care system perspective. The participants appreciated being engaged in identifying and discussing concerns on safety and capacity early on; the richness of the discussions and the findings highlighted the usefulness of early stakeholder engagement when developing implementation strategies for new drugs and regimens. Studies using similar methods could be relevant to conduct future implementation research for a variety of novel drug interventions. A broader perspective should be obtained by also assessing patient perspectives. This limitation notwithstanding, our study provides some baseline evidence that could be useful for the conduct of more robust studies on this, and related research topics.Our assessment provides rich information from different stakeholder groups on perceptions and expectations on the BPaL regimen. It shows that the adoption of BPaL in three high DR-TB burden countries will be accepted and feasible. BPaL could increase the treatment success rate and alleviate the individual and health care system burden of DR-TB. Active TB drug-safety monitoring and management is crucial to address concerns on linezolid safety. National strategic plans should incorporate both the identified and anticipated country-specific challenges and requirements to facilitate smooth roll out of the BPaL regimen.Additional file 1.Additional file 2.Additional file 3.


Japan is the world’s fastest ageing population, with a higher prevalence of dementia than in the UK. Less clear is the role of socioeconomic inequalities in neurocognitive disorders between these countries. This study aims to assess comparatively the relationship between education, a marker of cognitive reserve, and income in relation to mild cognitive impairment (MCI) and dementia in England and Japan. We ascertained MCI using a validated algorithm based on one standard deviation below the mean on two standardised cognitive tests. Multinomial logistic regression models were used to study the associations between socioeconomic markers and MCI/dementia. The prevalence of MCI was almost twice as high among English adults compared to Japanese. Results suggest that nations are similar in overall socioeconomic inequalities of MCI/dementia, but this might differ across socioeconomic markers. Considerable variability in the health inequalities could be attributed to the country-specific socio-cultural-political factors, which remains to be further explored.


Brain network models derived from graph theory have the potential to guide functional neurosurgery, and to improve rates of post-operative seizure freedom for patients with epilepsy. A barrier to applying these models clinically is that intracranial EEG electrode implantation strategies vary by centre, region and country, from cortical grid & strip electrodes (Electrocorticography), to purely stereotactic depth electrodes (Stereo EEG), to a mixture of both. To determine whether models derived from one type of study are broadly applicable to others, we investigate the differences in brain networks mapped by electrocorticography and stereo EEG in a cohort of patients who underwent surgery for temporal lobe epilepsy and achieved a favourable outcome. We show that networks derived from electrocorticography and stereo EEG define distinct relationships between resected and spared tissue, which may be driven by sampling bias of temporal depth electrodes in patients with predominantly cortical grids. We propose a method of correcting for the effect of internodal distance that is specific to electrode type and explore how additional methods for spatially correcting for sampling bias affect network models. Ultimately, we find that smaller surgical targets tend to have lower connectivity with respect to the surrounding network, challenging notions that abnormal connectivity in the epileptogenic zone is typically high. Our findings suggest that effectively applying computational models to localize epileptic networks requires accounting for the effects of spatial sampling, particularly when analysing both electrocorticography and stereo EEG recordings in the same cohort, and that future network studies of epilepsy surgery should also account for differences in focality between resection and ablation. We propose that these findings are broadly relevant to intracranial EEG network modelling in epilepsy and an important step in translating them clinically into patient care. Bernabei et al. report that electrocorticography and stereo EEG provide different quantifications of epileptogenic zone connectivity due to differences in electrode type and implant patterns. After correcting for sampling differences between modalities, they find that more focal forms of epilepsy surgery target regions of weaker connectivity compared to the remaining epileptic network.  From the earliest days of intracranial EEG (iEEG), two major approaches have been used by clinicians for these purposes. In North America, Penfield’s use of subdural grid and strip electrodes for electrocorticography (ECoG) persists at many major centres now decades after its initial use.,,,,,,,Recent evidence supports the hypothesis that epilepsy arises from disordered connectivity,While networks derived from functional MRI and diffusion imaging easily generalize across patients and centres due to congruence in their full-brain spatial sampling, iEEG functional networks suffer from sparse sampling and implant heterogeneity. Still, the problem of spatial sampling bias which affects iEEG networksThere are many differences in implantation strategy between ECoG and SEEG that arise from the electrode hardware itself. Patients implanted with a large ECoG grid will have regular spacing between contacts in the same electrode (e.g. an 8×8 contact grid), supplemented with a few additional strip and depth electrodes in other regions, as needed. The implantation strategy is much more heterogeneous in SEEG. This heterogeneity can manifest itself in the following ways: (i) a wide range in the number of depth electrodes & electrode contacts from centre to centre; (ii) different spatial orientations and density of depth electrode implantation; (iii) different assortments of anatomical targets, electrode spacing; and (iv) different levels of implant bilaterality.In this study, we explore the effect of implant strategy in a retrospective cohort of patients with drug-resistant temporal lobe epilepsy who were evaluated with either ECoG or SEEG for invasive treatment. We hypothesize the following: (i) ECoG and SEEG networks have distinct properties due to different patterns of spatial sampling, and (ii) differences in network properties between ECoG and SEEG will impact the observed relationship between resected and spared tissue. We aim for our findings to help translate personalized network models of epilepsy into clinical practice, and to inform other applications of iEEG connectivity analysis.,We retrospectively analysed a data set consisting of 33 patients who underwent intracranial recording during evaluation for epilepsy surgery at the Hospital of the University of Pennsylvania (HUP). Sixteen of these patients had implants with grid, strip, and a small number of depth electrodes, while the remaining patients had only stereotactically placed depth electrodes. In this study, we refer to cortical-predominant patients as the ‘ECoG’ group while patients with only depth electrodes constitute the ‘SEEG’ group. All patients underwent either resection or laser ablation after electrode explant, however, in subsequent sections we use the term ‘resected’ tissue to include ablation patients as well. We chose only patients that achieved good outcome, assessed at 6 months post-operatively, to maximize the likelihood that tissue removed in surgery contained the epileptogenic zone. ,Each patient underwent a standard epilepsy imaging protocol including pre-implant MRI, post-implant CT & MRI, and post-resection MRI. We have previously described our method for localizing electrode locations in detail,,,We calculated functional connectivity using a pipeline that we have previously described and validated.ijA is link between nodes i and j, k are edge weights, m is the sum of all edge weights in the graph, and To probe how network structure differs between ECoG and SEEG, we detected communities using modularity maximization, which labels nodes so that each community consists of nodes that are more connected to each other and relatively less connected to all other nodes outside of their community.in-vivo, we compared the ability of connectivity derived from these modalities to distinguish resected and spared tissue. We chose the simple network metric of node strength, computed as the sum of all edge weights connecting it to all other nodes and is computed as is is the strength of node i, ijA is the adjacency matrix element containing the edge weight between node j and node i, and N is the number of nodes. Our group and others have previously demonstrated that high node strength localizes the epileptogenic zone and predicts surgical outcome in patients implanted with ECoG,,To illustrate the importance of differences in the way ECoG and SEEG represent epileptic networks rs) which quantifies the area under the receiver operating characteristic curve for classifying nodes as either resected or non-resected.rs has been previously studied and validated for its ability to quantify whether networks have sufficient information to determine resected or non-resected regions.,In our comparisons of network properties between ECoG and SEEG groups, we primarily used nonparametric statistical tests such as the Wilcoxon rank-sum test, as they do not assume that data are normally distributed. To assess the ability for node strength to detect epileptogenic regions, we used a metric known as the distinguishability statistic , many depth electrodes localized to white matter and ultimately the number of nodes in grey matter (GM) across ECoG and SEEG was similar . The median number of contralateral nodes was higher in SEEG than ECoG (25 vs. 4), however, this did not reach statistical significance (P = 0.06). Despite the modest differences in hemispheric differences in GM nodes, we observed similar median internodal distances between ECoG and SEEG . Overall, the targets sampled by ECoG and SEEG for patients with temporal lobe epilepsy were similar, implying that differences in anatomy and internodal distance alone would not primarily drive any subsequent differences in network models.We first asked what differences in the location and extent of anatomic sampling exist between ECoG and SEEG. After quantifying the top 15 anatomical targets implanted by each approach , we founrs. This value is high when resected nodes are weaker than the spared network and low when they are stronger , which was unexpected given that all patients had temporal lobe epilepsy and achieved good surgical outcome (P < 0.01), however, resected depth electrodes were not higher in strength than non-resected depth electrodes . Furthermore, non-resected depth electrodes were higher in strength than non-resected surface electrodes , and resected depth electrodes were higher in strength than resected surface electrodes . These findings, and the sizable proportion of resected depth electrode contacts in ECoG could account for the observed difference in Drs values between the two implantation strategies.Although ECoG and SEEG sample from similar brain regions, they may not represent the epileptogenic regions similarly from a network perspective. We aimed to gauge the ability of each implant strategy to distinguish resected and spared tissue using the Dstronger . Across  outcome . We then outcome . In thesrs differences between SEEG and ECoG by regressing for internodal distance in an electrode-specific manner. We fit a nonlinear regression model to ECoG and SEEG separately (P < 0.01). Furthermore, we calculated modularity in distance-corrected networks (P < 0.01). Analogous results for broadband cross-correlation and low-gamma coherence are found in We sought next to determine whether we could adequately correct for our findings of Dparately  using a parately , represenetworks  and founWe finally asked how our internodal distance correction would affect resection zone distinguishability in ECoG and SEEG, and whether we could correct for any remaining differences in network localization by simplifying initial networks to reduce sampling differences. Owing to the different balance of ipsilateral and contralateral nodes in ECoG and SEEG  and the rs for each of the three modifications: (i) correction for internodal distance (DC); (ii) using only unilateral nodes ipsilateral to the resection zone (UL); and (iii) atlas-level ROI (AR) . Analogous results for broadband cross-correlation and low-gamma coherence are found in rs values compared to ECoG even for this condition. As ECoG and SEEG networks with distance-corrected, unilateral, ROI-level nodes should be as similar as possible, we hypothesized that remaining differences were a result of differences in the extent of surgical intervention between groups  . For eacn groups . Indeed,Understanding the sampling differences of different implant approaches is critically important when applying network models to interpret iEEG data. Here, we showed how the unique characteristics of ECoG and SEEG sampling result in distinct properties of derived networks despite similar clinical targets. Node strength, a frequently studied network metric in epilepsy, had an unpredictable relationship between resected and spared tissue, and accounting for internodal distance & electrode type still resulted in distinct network properties. We also establish that these general patterns are present in different frequency bands and in both coherence and correlation measures of functional connectivity. Finally, we provided several methods to partially mitigate the effects of sampling bias introduced by implantation strategy on network models, and showed that remaining differences are associated with the focality of the subsequent resection or ablation.,a priori suspicions present before implant.Our study adds to the growing body of literature on methodological considerations for applying network models clinically.,While the finding that ECoG and SEEG have distinct network connectivity patterns in resected versus spared tissue is significant, it may in part reflect conceptual differences underlying these implant strategies. ECoG attempts to map the boundaries of epileptogenic cortical regions by assessing seizures and interictal activity,While others have reported differences in connectivity values between depth electrodes and surface electrodes,rs. On the other hand, if the true epileptogenic zone is in the hemisphere with fewer electrodes, network models may struggle with localizing it. Furthermore, our finding that averaging edges between pairs of brain regions to convert nodes to atlas ROIs maintains similar performance implies that network models may not need dense sampling within regions, but instead may benefit from sampling inter-regional connections.The results of our sampling correction process  reveal irs and number of regions targeted may imply that the truest representation of the epileptogenic zone is of low node strength relative to the rest of the brain. This notion is supported by studies demonstrating cellular loss in these regions, particularly the hippocampus, in temporal lobe epilepsy.,Another important consideration to the application of network models is the issue of surgical focality which may differ significantly between resection and ablation patients. While many previous studies focus on resection patients, which may extend to natural anatomic boundaries therefore removing additional, non-epileptogenic tissue, a large part of our cohort underwent ablation in which lesions are relatively small and more specific to the epileptogenic zone. Indeed, others have found that a large number of nodes within and outside of the resection zone is important for accurate outcome prediction from interictal connectivity, which may be impossible for ablation patients. In this context, our finding of a negative correlation between DDespite its encouraging results in illuminating the differences between ECoG and SEEG networks, our study has several key limitations. We focussed our analysis on node strength, one of the simplest graph theory metrics that has been studied frequently in epilepsy, so this measure may not fully represent the complexity of abnormal networks in this disorder. However, given that node strength is among the least sensitive network metrics to sampling bias,Ultimately, clinical judgement of risk and reward will drive the choice between ECoG and SEEG for individual patients. While the path of clinical translation for network models is complicated by inherent sampling biases across techniques and varying surgical practice among institutions, we believe it is vital to compare and contrast studies of ECoG and SEEG and recognizing that each provides a distinct view of the brain’s underlying connectivity but that neither are ‘correct’. Finally, we believe that carefully understanding the sampling properties of networks mapped by iEEG can extend the use of graph theory to broader problems in translational human neuroscience.Brain Communications online.fcab156_Supplementary_DataClick here for additional data file.


Retinal painting, anorthoscopic perception and amodal completion are terms to describevisual phenomena that highlight the spatiotemporal integrative mechanisms that underlieprimate vision. Although commonly studied using simplified lab-friendly stimuli presentedon a computer screen, this is a report of observations made in a novel real-world contextthat highlight the rich contributions the mechanisms underlying these phenomena make tonaturalistic vision. Retinal painting, anorthoscopic perception and amodal completion are terms to describevisual phenomena that highlight the spatiotemporal integrative mechanisms that underlieprimate vision. Vision scientists have spent over a century studying the mechanismsunderlying these phenomena through the use of behavioural  andmoreShown in At slow walking speeds, this window has a darkened transparency and takes on a flickeringcharacteristic not unlike an old movie played slowly, as the persistent high contrast imageprovided by each gap overlaps the intervening boards. At running speeds or when looking outa car window, which also provides a more stable viewing platform, the flicker is muchfaster, less intrusive and the image appears brighter, remarkably uniform and stable. Thisobservation is consistent with the Talbot-Plateau Law which asserts the perceived intensityof a flashing light will be determined by the ratio of the on and off periods  and woulFor reasons that I suspect have to do as much with my own incompetence as well as depth offocus, exposure duration and discrete sampling, my attempts to video the effect have provedvery ineffective. Demonstration Video 1 provides glimpses of the scene down river from thebridge; although the video completely fails to capture the experience of viewing in person.Winter in Vermont is cold and there is a certain joy that comes with the first signs ofwarming weather, melting snow and the transition to what locals call “mud season”. Dirtroads covered in ice and snow for months turn into rutted, muddy affairs dotted withpuddles. As I continued my walk beyond the covered bridge, I found myself on one suchpuddle-filled dirt road. When standing still, within the puddles small glimpses can be seenof the reflections of the overhanging branches of the trees that line the road .Taken as a single snapshot moment in time, these glimpses provide very little sense ofcanopy above. This is likely because the piecemeal features reflected in each puddlecomprise distinct complex textures with little between-puddle collinearity  or 3D coIn conclusion, I appreciate the light-hearted nature of visual illusions and the simpledemonstrations of perceptual phenomena highlighted in our textbooks, deployed on ourvision-laboratory testing computers and sent to us via email by friends and relatives. Thatsaid, there is something special about the genuine feelings of happiness when theintricacies of visual perception are revealed to us through real-world experiences likethose described here, particularly coming amidst these times of pandemic tragedy. Isincerely hope the reader will enjoy this account of the spatiotemporal integrativemechanisms that underly our visual experiences as much as I have.


The cephalopod arm armature is certainly one of the most important morphological innovations responsible for the evolutionary success of the Cephalopoda. New palaeontological discoveries in the recent past afford to review and reassess origin and homology of suckers, sucker rings, hooks, and cirri. Since a priori character state reconstructions are still ambiguous, we suggest and discuss three different evolutionary scenarios. Each of them is based on the following assumptions: (1) Neocoleoidea uniting extant Decabrachia and Octobrachia is monophyletic (= proostracum-bearing coleoids); (2) extinct Belemnitida and Diplobelida are stem decabrachians; (3) proostracum-less coleoids  represent stem-neocoleoids; (4) Ammonoidea and Bactritoidea are stem coleoids. We consider a scenario where belemnoid hooks derived from primitive suckers as well-supported. Regarding belemnoid hooks and suckers as homologues implies that belemnoid, oegopsid, and probably ammonoid arm hooks arose through parallel evolution. Our conclusions challenge the widespread opinion, whereupon belemnoid hooks evolved de novo, and instead support earlier ideas formulated by Sigurd von Boletzky. Chitins have so far been analysed only in gladiuses, beaks, and in the sucker surfaces of octopods. At least, in hematoxylin and eosin-stained sections of post-embryonal Sepia, both the sucker ring and the adjacent infundibulum exhibit the same colour  or imprinted in finely laminated sediments , the diameter of the ring aperture merely exceeds 0.01 mm  oegopsid Dosidicus gigas (Oegopsida: Ommastrephidae).The idea of “chitinous” sucker rings is widespread , and the Golden cuttlefish Sepia esculenta (Sepiida: Sepiidae). Apart from its well-balanced mixture of stress and strain properties, further intriguing physicochemical and thermomechanical characteristics of suckerin attracted the interests of material scientists; in particular their high elastic modulus combined with thermoplastic behaviour appears to be highly remarkable . These circular structures have later been re-interpreted as the attachment ring (infundibulum) of belemnoid suckers as very similar imprints associated with Acanthoteuthis from the Solnhofen plattenkalks suggest . Instead, Gramadella is more likely a gladius-bearing octobrachian similar to Proteroctopus that is evidently missing sucker rings  possess a single, significantly enlarged, sabre-like hook on each of their dorsolateral arms  are equipped with claw-like structures Fig. c, d. TheGonatus fabricii have been compared and tracked. Despite some problematic interpretations, he ascertained that the hook is a derivate of the ring wall rather than from the infundibulum (= Kristensen’s attachment ring). The same author also remarked on page 113 that “Transitional stages […] were impossible to find, so the development between the two stages is probably very rapid.”Although nineteenth century workers such as Owen , p. 81 wDespite a certain similarity in form and development in various divisions , profound differences in hook formation can be found” [translated from German]. Pfeffer . They differ from the Paratethys specimens particularly in having a tip that is curved towards the base, i.e. forming an uncinus that is typical for oegopsid hooks Fig. f. In botHooks superficially similar to oegopsid hooks also furnished the oral surfaces of the arms of belemnoid coleoids  arranged, present on each of the ten arms, and evidenced for at least members of the Phragmoteuthida and Belemnitida (assumed for Aulacoceratida and Diplobelida). Mega-hooks  and mega-hooks (> 5 mm). Micro-hooks are abundant, variable in shape, biserially  come from the Carboniferous Francis Creek shale; the last unambiguous belemnoid hooks occur in Maastrichtian deposits , many authors agreed that belemnoid hooks did not develop through rings .Belemnoid hooks are either preserved as empty imprints or, if substantially conserved, often carbonised  cirri are unlikely homologues of belemnoid hooks”.Strand- or filament-like cirri Fig. a, b are Plesioteuthis from the Late Jurassic Solnhofen Plattenkalks and Glyphiteuthis from the Late Cretaceous Lebanon Plattenkalks; Bandel & Leich, Plesioteuthis from the Kimmeridgian of Painten, the dark organic preservation of cirri suggests a light (rudimentary?) sclerotisation homology of cephalopod hooks is given in Table In contrast to octobrachian and decabrachian suckers, whose homologous nature was never questioned seriously, the occurrence and homologue nature of arm hooks has created some confusion in the literature. This confusion has its roots deep in pioneering coleoid research. Münster , for insVoltz  like manAs far as arms are sufficiently well preserved in the fossil record of belemnoids, …,these arms bear structures resembling the hooks of recent teuthoids; they can only be interpreted in analogy to the latter.” Jeletzky  and the Neocoleoidea is no more tenable, because the autapomorphies previously used to establish both taxa are questionable . The taxon Neocoleoidea is monophyletic only after the inclusion of proostracum-bearing belemnoids  with Phragmoteuthida containing the last common ancestors of the Decabrachia and Octobrachia , the phylogenetic bracket generally opens the possibility that primitive sucker cups evolved in the coleoid stem lineage. If suckers evolved later in the stem lineage of the Neocoleoidea, a morphogenetic program for suckers did not exist in stem coleoids (Ammonoidea and Bactritoidea). Carboniferous Donovaniconida and Hematitida might have lacked suckers as well. Primitive suckers then possibly evolved in early aulacoceratids (Permian to Jurassic) or phragmoteuthids . Such a late development would challenge the idea whereupon suckers derived from transverse bulges present in the nautiloid lineage  ancestors or the decabrachian type of sucker ring represents a rudimentary belemnoid hook. A hypothetical transformation from an abnormal (rudimentary) belemnoid hook type to the decabrachian type of sucker ring is based on Late Cretaceous belemnoid hooks typified by a wide basal opening Fig. i.Anyway, regarding belemnoid hooks and decabrachian sucker rings as homologues implies that belemnoid hooks were likewise made of suckerin. The main component of the decabrachian sucker rings would have then originated along with the first appearance of belemnoid hooks during the Carboniferous in stem neocoleoids at the latest.In this scenario, oegopsid and belemnoid hooks automatically represent parallel developments (= independent evolution of homologous characters) as indirectly advocated by Naef . If ammoIf belemnoid hooks evolved from primitive suckers, but independently from sucker rings, they must have arisen from an unknown sucker structure. It is possible that belemnoid hooks represent outgrowths of the infundibulum by contrast to sucker rings, which develop from the inner (acetabular) sucker wall. Belemnoid hooks were consequently composed of a material other than suckerin or chitin.Ceratisepia), as the oldest unambiguous evidence of crown decabrachians, were reinforced with rigid rings. Such a comparatively late origin of sucker rings suggests that they evolved in a hook-less belemnoid subgroup  as less likely, whereas scenario 1 (belemnoid hooks derived from primitive suckers) is—contrary to the current opinion and in agreement with Naef , JeletzkHow can juvenile sucker rings with a tiny diameter develop into a ring with a larger diameter? How is it possible to increase the diameter of a stiff sclerotised structure? Stretching of the ring wall might be one option, but the ring wall is said to grow only in thickness (from the inside outward) and not by adding intermediate material. Resorption of the inner ring wall might be another option to increase the diameter, but such a pattern would afford an appropriate epithelium, which apparently does not exist.What happened to the remaining teeth? Are they resorbed or covered with additional material?Our conclusion may be verified by new insights about the growth of decabrachian rings and hooks, which is not yet fully understood. The absence of transitional stages between a typical multi-toothed sucker ring and a hook morphologically close to the adult hook as well as the assumption of a very rapid development might alternatively point to periodical shedding and the formation of new larger rings and hooks—similar to the new formation of the octopod infundibulum lining. However, this is apparently not the case. As a result of this, the following questions about the mode of sucker ring growth are still open:What was the original composition of belemnoid hooks?Is carbonisation more likely in purely proteinous or chitinous structures?Similar questions concern the formation of belemnoid hooks. Two difficult questions are crucial for the assessment of belemnoid hooks:Both questions concern the general mode of formation: each of the structures under discussion probably share the absence of continuous growth .The lack of substantially preserved belemnoid arm hooks in localities  with a demonstrably good preservation of originally chitinous structures as well as the fact that oegopsid hooks do not contain chitin (see above) let a chitinous composition of belemnoid arm hooks appear improbable. Instead, we are inclined to consider an interplay of structural proteins such as suckerin as it is the case in oegopsid hooks.