Data are limited on the frequency of ‘consensus decisions’ between sub-specialists attending a neurovascular multidisciplinary meeting (MDM) regarding management of patients with extracranial carotid/vertebral stenoses and post-MDM ‘adherence’ to such advice.This prospective audit/quality improvement project collated prospectively-recorded data from a weekly Neurovascular/Stroke Centre MDM documenting the proportion of extracranial carotid/vertebral stenosis patients in whom ‘consensus management decisions’ were reached by neurologists, vascular surgeons, stroke physicians-geriatricians and neuroradiologists. Adherence to MDM advice was analysed in asymptomatic carotid stenosis (ACS), symptomatic carotid stenosis (SCS), ‘indeterminate symptomatic status stenosis’ (ISS) and vertebral artery stenosis (VAS) patients, including intervals between index event to MDM + / − intervention.N = 26), with a median interval from MDM to revascularisation of 5.5 days (IQR: 1–7 days). Thirty patients underwent revascularisation. Two out of twenty-nine patients (6.9%) with either SCS or ISS had a peri-procedural ipsilateral ischaemic stroke, with no further strokes/deaths during 3-months follow-up.One hundred fifteen patients were discussed: 108 with carotid stenosis and 7 with VAS. Consensus regarding management was noted in 96.5% (111/115): 100% with ACS and VAS, 96.2% with SCS and 92.9% with ISS. Adherence to MDM management advice was 96.4% (107/111): 100% in ACS, ISS and VAS patients; 92% (46/50) in SCS patients. The median interval from index symptoms to revascularisation in 50–99% SCS patients was 12.5 days (IQR: 9–18.3 days; The high frequency of inter-specialty consensus regarding management and adherence to proposed treatment supports a collaborative/multidisciplinary model of care in patients with extracranial arterial stenoses. Service development should aim to shorten times between MDM discussion-intervention and optimise prevention of stroke/death. It hpatients . The ESV 60% ACS –10. Howe1. Assess the proportion of patients in whom a consensus management decision was reached by the attending surgeons and physicians from different specialities.2. Assess the ‘adherence’ to the advice offered regarding treatment by the attending physician/surgeon after they had further discussions with the patient following the MDM.3. Document the categories of medical or interventional advice provided to patients with extracranial carotid or vertebral artery stenosis at a neurovascular MDM.4. Assess the time from symptom onset to MDM discussion and the time from MDM discussion to intervention, as appropriate.5. Assess short- and medium-term outcomes in our cohort following MDM discussion.The aims of this multi-centre audit and quality assessment and improvement process were the following:1. Consensus decisions regarding management would be reached in the majority of patients.2. Most treating physicians/surgeons and patients would ‘adhere’ to the primary treatment advice offered at the neurovascular MDM.3. The majority of SCS patients would have revascularisation and the majority of ACS and vertebral artery stenosis (VAS) patients would have optimal medical treatment as their primary recommended treatment.4. Our practice would be in keeping the international guidelines regarding optimal time from symptom onset and MDM discussion + / − intervention, where necessary.5. Outcome event rates before discharge and during medium-term follow-up would be in keeping with the recommended acceptable thresholds suggested by international guidelines.We planned to compare our practice and outcomes against recommendations of the ESO 2021 guidelines  and the We hypothesised the following:Data from a well-established, weekly neurovascular MDM at our university teaching hospital, a secondary and tertiary regional referral centre for neurovascular patients, were prospectively collected between 21st of Sept 2017 and 27th of Feb 2020  for this audit and quality improvement (QI) project. The meeting is run in close collaboration with our attending consultant neurologists, consultant vascular surgeons, consultants in older adult/stroke medicine and consultant diagnostic neuroradiologists. At this meeting, the attending consultants have the opportunity to discuss any patients with extracranial carotid and/or vertebral artery stenosis who may be suitable for revascularisation with endarterectomy or endovascular treatment, or in whom they feel optimal medical therapy may be the best treatment option. If very urgent treatment decisions regarding endarterectomy or endovascular treatment need to be made in clinically unstable patients prior to the weekly neurovascular MDM, the guideline at our hospital is that such cases must be discussed by a consultant vascular surgeon with either a consultant neurologist or stroke physician to reach consensus between the two consultants in question before proceeding to intervention. This audit/quality assessment and improvement process were coordinated by the chair of the neurovascular MDM  and a consultant vascular surgeon .2 scores in TIA patients (range: 0–7) and modified Rankin scale scores in living patients who had experienced an ischaemic stroke (range: 0–5).The data were entered prospectively onto a standardised MDM proforma, which included a summary of the clinical history, known prescribed medications and key examination findings, neurovascular investigations, the treating clinician’s final decision regarding the symptomatic status of the artery(ies) in question, ABCDFor the purpose of this study, patients were included in the ACS group if they were incidentally noted to have extracranial internal carotid stenosis on vascular imaging which had not caused any relevant neurovascular presenting symptoms prior to MDM discussion; the ACS was typically noted on colour Doppler ultrasound and confirmed on computed tomography angiography (CTA) or magnetic resonance angiography (MRA) in the majority of cases. Patients were included in the symptomatic carotid stenosis group if they had experienced a TIA or ischaemic stroke in the vascular territory supplied by an ipsilateral carotid artery stenosis of any severity which prompted MDM discussion, with no other cause for their symptoms identified. Patients were defined as having carotid stenosis of ‘indeterminate symptomatic status (ISS)’ if it was unclear on clinical grounds whether the ipsilateral carotid artery (ICA) stenosis had caused their neurovascular symptoms or if they had more than one potential cause for their TIA/stroke in the vascular territory supplied by an ICA stenosis . Patients were defined as having VAS if they had a TIA or ischaemic stroke in the vascular territory supplied by a stenosed extracranial vertebral artery on either side.The severity of arterial stenosis using extracranial computed tomography angiography (CTA) or magnetic resonance angiography (MRA) was formally quantified on the day of the MDM by a neuroradiologist (or by the chairing vascular neurologist in his/her absence (DJHM)) according to the North American Symptomatic Carotid Endarterectomy Trial (NASCET) method of estimating stenosis . Stenoseprimary composite clinical outcome’ of peri-procedural/in-hospital stroke or death before discharge, as well as other pre-specified peri-procedural complications documented by the treating physician/surgeon. These included recurrent ipsilateral/contralateral/territorial TIA; and recurrent ipsilateral/contralateral/territorial ischaemic or haemorrhagic stroke. We also analysed available data on pre-specified ‘secondary clinical outcomes’ which had been recorded by the clinical teams during routine clinical follow-up, and which were either listed on the MDM proforma, and/or recorded in the hospital notes or electronic case records: stroke or death alone during follow-up between discharge and the 3-month follow-up visit; transient ischaemic attack, stroke or death between discharge and the 3 month follow-up visit in SCS patients, ISS and ACS patients; recurrent ipsilateral or contralateral territorial TIA; confirmed MI/coronary revascularisation (stenting/CABG); vascular death/other causes of death; consultant-confirmed disabling cranial nerve palsy/nerve injury; significant access-site haematoma; confirmed hyperperfusion syndrome; or other causes of death between discharge and the 3-month follow-up visit. If patients were only followed up at a referring centre after discharge, their attending consultant stroke physician was contacted to enquire about the occurrence of any of the above outcome events after the MDM.It was the practice at our centre during the audit period that all patients were assessed by an attending neurologist or stroke physician before and after any revascularisation procedure had been performed by our vascular surgeons to independently identify any peri-procedural outcomes; relevant findings were recorded in the hospital notes. We collated all available contemporaneously recorded data from the hospital notes and/or electronic case records on our server to assess the frequency of the ‘The time intervals from symptom onset to MDM discussion, and from MDM discussion to intervention in appropriate cases, were calculated to determine whether we were treating patients in line with best international practice according to recent clinical practice guidelines . All datBecause these data were collated as part of a prospective audit and quality assessment and improvement programme, our Local Research Ethics Committee (LREC) hospital guidelines indicate that formal LREC approval for such a study is not required. However, the study was registered with the Tallaght University Hospital-AMNCH clinical audit committee, all data were securely stored in electronic format, and all presented data were fully pseudonymised so that no individual patient could be identified from the data contained within this manuscript. The study and analyses were conducted in accordance with best ethical practice and supervised by an experienced consultant who is International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use Good Clinical Practice (ICH GCP)-certified.t tests were used for comparison of paired and unpaired parametric variables, respectively. The Wilcoxon signed rank test and the Wilcoxon rank sum test were used for comparison of paired and unpaired non-parametric variables, and the Kruskal–Wallis rank sum test was used for comparison of multiple non-parametric variables, where appropriate. Chi-squared or Fisher exact tests were used to compare proportions between groups, where appropriate. All statistical calculations were performed using SPSS®  and PRISM 9 software (GraphPad Software Inc).Paired or unpaired One hundred and fifteen patients were discussed at our weekly neurovascular MDM over a 29-month period (21/09/2017–27/02/2020): 108 with carotid stenosis and 7 with VAS. Demographic data are displayed in Table Full adherence to advice provided at the MDM was observed in 92% (46/50) of SCS patients, and in all with ACS, ISS and VAS. Three patients in whom revascularisation was recommended did not proceed to intervention and were managed by OMT; one patient who was advised to have OMT was rediscussed and then had further investigations, but did not subsequently undergo urgent revascularisation because the treating vascular surgeon did not feel it was safe to proceed Table .Table 3Revascularisation was recommended in 27/52 (51.9%) patients with SCS, with OMT in 20 (38.5%), and further investigations or rediscussion recommended in 5 (9.6%) of these patients. Revascularisation was only initially recommended in 1/28 (3.6%) patients with ISS who had a ≥ 70–99% ICA stenosis, but in no patients initially with ACS or VAS (Table N = 26), with a median interval from MDM discussion to intervention in these SCS patients of 5.5 days , and from MDM to intervention was 1 day (IQR: 1–4.5 days). One patient with a symptomatic left internal carotid artery (LICA) occlusion and minor ischaemic stroke, who also had 80% right internal carotid artery (RICA) ACS, subsequently underwent deferred right CEA 2.5 months after MDM re-discussion, completion of further investigations and after recovery from a LICA territory stroke, so the time interval from MDM to intervention was 75 days.Thirty patients had revascularisation; 28 patients underwent revascularisation following a consensus management decision at the MDM, and the remaining 2 patients were subsequently advised to have revascularisation after the MDM. The median interval from symptom onset to MDM discussion in patients with 50–99% SCS who subsequently underwent revascularisation was 9 days (IQR: 7–12.75). The median interval from index symptoms to intervention in patients with 50–99% SCS was 12.5 days  who underwent revascularisation, or two of 29 patients with either SCS or ISS (6.9%) who underwent revascularisation had a peri-procedural primary clinical outcome of ipsilateral ischaemic stroke Table . The firTwo of twenty-six SCS patients (7.6%) had complex presentations with their stroke, one of whom had haemorrhagic transformation of a large ipsilateral infarct resulting in a delay to revascularisation with CEA until 61 days after index symptom onset . The other patient with LICA SCS presented with a left MCA territory ischaemic stroke with right sided weakness and sensory inattention. The patient was initially treated with aspirin monotherapy by his treating physician, but deteriorated overnight with embolisation to the ipsilateral left MCA causing aphasia prompting transfer to the National Thrombectomy Centre for urgent MCA mechanical thrombectomy and LICA stenting the following day without clinical improvement. Because no initial haemorrhagic transformation was noted on repeat imaging, clopidogrel was added to aspirin therapy. This patient, who was the only one in this series treated with urgent stenting, subsequently developed delayed symptomatic haemorrhagic transformation of a left hemispheric infarct on aspirin + clopidogrel combination therapy and prophylactic subcutaneous enoxaparin 17 days after admission, requiring rationalisation of antithrombotic therapy.contralateral cerebrovascular events during our follow-up period, but all were treated with modifications of their medical therapy regimens, and none underwent revascularisation after assessment by their treating physicians or surgeons. One of 24 ISS patients (4.2%) in whom follow-up data were available had a confirmed ST-elevation myocardial infarction requiring coronary stenting and a change in his antiplatelet therapy from aspirin + dipyridamole MR to aspirin + clopidogrel combination therapy.One hundred and two of 115 patients (88.7%) with carotid or vertebral artery stenosis had complete 3-month follow-up data available Table . There wThis prospective audit/QI project has shown the important finding that a neurovascular MDM can enable colleagues in clinical practice in neurology/stroke medicine, vascular surgery and neuroradiology to openly discuss and reach a consensus decision regarding evidence-based management in the vast majority 96.5%) of patients with extracranial carotid or vertebral stenoses. This should encourage the establishment of a neurovascular MDM at Neurovascular/Stroke Centres where such a service does not exist, as advised in recent clinical practice guidelines , 5, 6, w% of patibefore initial discussion at the MDM in the two studies, because we less frequently recommended doing further investigations in ACS or SCS patients than Rimmele et al.Furthermore, most treating physicians/surgeons (96.4%) adhered to the main treatment advice offered at the neurovascular MDM. This is similar to the level of adherence seen in SCS patients (94%), but higher than the level of adherence observed in ACS patients (69%) in a prior study by Rimmele et al. . Our higRevascularisation was recommended in the majority of SCS patients at our neurovascular MDM 51.9%), in keeping with international guidelines , 5, 6 an1.9%, in In contrast, all patients with ACS and VAS were initially advised to have OMT following discussion at our MDM. Only one of 22 ACS patients (4.5%) with moderate restenosis had a recurrent haemodynamic TIA during follow-up which the attending physician opted to treat with alteration of optimal medical therapy. Therefore, the proportion of ACS patients in whom revascularisation was ultimately recommended was much lower at our centre (0% at the MDM pending further investigations and 4.5% (1/22) after completion of investigations) than at a university medical centre in Germany (26.8% of cases) . Aside fIt is of interest that 75% of patients with ISS who had potential competing mechanisms responsible for their presenting symptoms, and whose management was discussed in this ‘real-world’ clinical setting at our Neurovascular centre, were also advised to have OMT by the various specialists in attendance Table . This suOur recommendation to revascularise the majority of SCS patients with CEA rather than with EVT is in keeping with recent clinical practice guidelines , 5, 6 anper se.Between 2005 and 2013, many vascular surgeons transitioned to performing most CEAs within 2 weeks of symptom onset, with the mean time to surgery decreasing from 25 to 6 days . The medThe observed risk of peri-operative in-hospital stroke following CEA was 7.6% in 50–99% SCS patients and 6.9% in the SCS and ISS subgroups combined, with no deaths. This risk is higher than the recently-proposed ‘recommended threshold’ of < 4% for the independently-assessed risk of in-hospital stroke/death , and onlpotential limitations. Neurological outcomes either in the peri-procedural period in patients undergoing revascularisation, or during follow-up in those who were treated with revascularisation or OMT were recorded by the primary treating neurologist/stroke physician and were not verified further by another ‘independent’ neurologist/stroke physician who was not involved in the patients’ care; limited subspecialty staffing on site and in our region precluded such an approach. However, based on the aforementioned practice of independent assessment of all patients with carotid stenosis before and after revascularisation by the attending neurologist/stroke physician who collaborated with the attending vascular surgeon to deliver care, we believe that this audit reflects the findings of a real-world practice with independent objective assessment of outcome measures [This prospective audit and QI project had some measures , 33. Furmeasures . FinallyThe high frequency of inter-specialty consensus regarding management and adherence to proposed treatment supports a collaborative, multidisciplinary model of care in patients with extracranial arterial stenoses. Future service development should aim to shorten the time interval between MDM discussion and revascularisation, where appropriate, to optimise primary and secondary prevention of TIA or stroke, and to minimise peri-procedural risks of stroke or death during carotid endarterectomy.


Brugada syndrome (BrS) is a primary electrical disease predisposing to ventricular tachyarrhythmias and sudden cardiac death . The optIn the Journal of Clinical Medicine Special Issue “New Frontiers in Electrocardiography, Cardiac Arrhythmias, and Arrhythmogenic Disorders”, Tsai et al.  describeNotably, Mizusawa et al.  reportedMoreover, in their paper, Tsai et al.  noted thThe association between fever and ECG pattern may be particularly important in light of evidence for inflammation in the right ventricular outflow tract of BrS patients, which may trigger ventricular arrhythmias in predisposed hearts .It should be mentioned that a fever-induced type 1 Brugada ECG pattern may be awarded 3 points in the proposed Modified Shanghai scoring system for the diagnosis of BrS, compared to 3.5 points for a spontaneous type 1 Brugada ECG pattern and 2 points for a sodium channel blocker-induced Brugada type 1 ECG pattern  . Moreovehttp://www.brugadadrugs.org (accessed on 24 April 2023) and treatment of fever with antipyretic drugs) of asymptomatic  patients with fever-induced type 1 Brugada ECG pattern without documented ventricular arrhythmia and with negative family history (both of BrS and sudden death < 45 years) as the most reasonable approach to clinical management.Thus, in line with current ESC guidelines , we cons


Muscles exhibiting signs of LMN dysfunction, as indicated by pathological spontaneous activity at rest and/or increased discharge rates of motor unit action potentials, were associated with more severe paresis in both the acute and chronic stages after SCI . Moreover, the severity of LMN dysfunction in the acute stage was also associated with a greater degree of paresis . While both muscles with and without signs of LMN dysfunction were capable of regaining strength over time, those without LMN dysfunctions had a higher potential to reach full strength. Muscles with signs of LMN dysfunction in the acute stage displayed increased amplitudes of motor unit action potentials with chronic-stage needle EMG, indicating reinnervation through peripheral collateral sprouting as compensatory mechanism . Thus, LMN dysfunction represents a relevant factor contributing to motor impairment and recovery in acute cervical SCI. Defined recovery mechanisms  may at least partially underlie spontaneous recovery in respective muscles. Therefore, assessment of LMN dysfunction could help refine prediction of motor recovery after SCI.Paresis after spinal cord injury (SCI) is caused by damage to upper and lower motoneurons (LMNs) and may differentially impact neurological recovery. This prospective monocentric longitudinal observational study investigated the extent and severity of LMN dysfunction and its impact on upper extremity motor recovery after acute cervical SCI. Pathological spontaneous activity at rest and/or increased discharge rates of motor unit action potentials recorded by needle electromyography (EMG) were taken as parameters for LMN dysfunction and its relation to the extent of myelopathy in the first available spine magnetic resonance imaging (MRI) was determined. Motor recovery was assessed by standardized neurological examination within the first four weeks (acute stage) and up to one year (chronic stage) after injury. Eighty-five muscles of 17 individuals with cervical SCI  and a median age of 54 (28–59) years were examined. The results showed that muscles with signs of LMN dysfunction peaked at the lesion center (Χ In this context, the relevance of lower motoneuron (LMN) dysfunction—widely known to occur after SCI—has yet to be determined.7Outcomes in cervical spinal cord injury (SCI) are heterogeneous.and LMN damage or dysfunction contribute to paresis to a varying extent.8 Routine clinical assessment of voluntary motor strength, however, which is conducted according to the International Standards for Neurological Classification of Spinal Cord Injury (ISNCSCI),9 does not allow discrimination between upper and LMN damage, particularly early after injury. Without structurally intact LMNs at and below the level of injury, spared long-axon motor tracts will not be able to maintain a functioning neural pathway to exert voluntary motor control.11Paresis as a result of SCI is primarily caused by long descending pathway disruption such as the corticospinal tract, commonly referred to as the upper motoneuron. In particular, in cervical and lumbar SCI, both upper motoneuron 12–19 None of these studies, however, focused on the spatial extent or impact of LMN dysfunction on motor recovery after acute cervical SCI. Only one study analyzed signs of LMN impairment in a subset of participants with acute SCI, applying nerve conduction studies of the median and ulnar nerves—an approach that does not investigate specific LMN pools representing defined segmental levels.15Cross-sectional investigations have already reported signs of LMN dysfunction in human SCI at and remote from the injury site during chronic stages.20 This requires confounding factors, such as peripheral nerve injuries or myopathies, to be ruled out. In the acute stage, the parameters “pathological spontaneous activity at rest” (PSA) and increased discharge rates of “motor unit action potentials” (MUAPs) can be used to indicate dysfunction or loss of motor units/LMNs. In contrast, during the chronic stage, high amplitudes of MUAPs are indicative of subsequent compensatory reinnervation  and can be detected after LMN damage.21–23To date, needle electromyography (nEMG) is considered the gold standard clinical routine assessment to detect signals of LMN dysfunction recorded from muscles that are innervated by corresponding LMN pools.25 In addition, experiences from relevant studies regarding functional electrical stimulation (FES)-based neuroprostheses and nerve transfer surgeries have confirmed that LMN dysfunction is a relevant limiting factor.26Studies reporting multi-segmental innervation of muscles in cervical and lumbar regions have already discussed the clinical implications of LMN damage after localized injuries to the spinal cord or individual nerve roots.27Hence, the mentioned aspects imply that LMN dysfunction may play a more important role in functional recovery after cervical SCI than previously assumed. It is therefore clinically relevant to investigate LMN dysfunction already at an acute stage after SCI and evaluate its impact on motor recovery over the course of time. The present observational study primarily aimed to address these questions. These findings will not only further the understanding of (motor) recovery processes during comprehensive SCI care but also help predict responsiveness to the innovative therapeutic strategies mentioned above, all of which rely on the integrity of LMNs.For these reasons, individuals with acute cervical SCI prospectively underwent standardized neurological assessments according to ISNCSCI, nerve conduction studies, and nEMG. In addition, nEMG findings related to LMN dysfunction were correlated retrospectively with myelopathy as depicted in early routine spine MRI. This approach aimed to evaluate early MRI as an additional clinically relevant readout for indications of structural damage and to further refine the estimation of the rostrocaudal extent and pattern of affected LMNs.28 An illustration of the course of the study and related examinations is given in This prospective monocentric longitudinal, observational cohort study was approved by the ethics committee of Heidelberg University (S-516-2011) and registered in the German Clinical Trials Registry (Registry-no. DRKS00006258). Before enrollment, written informed consent was obtained from all study participants. Patients were recruited from 2012 to 2016. The time schedule for clinical examinations was derived from the European Multicenter Study about SCI (EMSCI).28 Administered medication that could potentially interfere with motor recovery was recorded. Three individuals received baclofen, another three received opioids, and none received benzodiazepines.Individuals with traumatic or ischemic SCI of any severity (AIS A–D) were identified by consecutive sampling and considered eligible primarily if they presented a neurological level of injury (NLI) between C3–C8. Because of a high number of screening failures , patient30 Participants were examined in accordance with the EMSCI time schedule at least once in the acute stage (16–40 days) and once in the chronic stage (150–186 or 300–546 days) after SCI  level upper extremity sum motor score on both sides of the body, UEMS_EXAM for the actual examined below-(motor) level upper extremity sum motor score on both sides of the body at the acute stage, and LoMF for the relative loss of motor function.33 With one exception, only upper extremity key muscles according to ISNCSCI were chosen.9 Instead of the flexor digitorum profundus muscle, the abductor pollicis brevis muscle, representing the spinal segment C8, was investigated because of better accessibility for nEMG.34 The biceps brachii muscle (C5), the extensor carpi radialis longus muscle (C6), the triceps brachii muscle (C7), and the abductor digiti minimi muscle (T1) were additionally examined to provide a comprehensive mapping of the segmental innervation in the cervical spinal cord.Needle EMG was chosen to detect signs of LMN dysfunction and performed in five defined key muscles of the dominant arm (C5 to T1) at three different examination sites by two board-certified neurologists.Care was taken to rule out other causes of nEMG abnormalities such as neuromuscular diseases, polyneuropathy, brachial plexus injury, and any other known lesion of the peripheral nervous system : (a) pas21–23 To eliminate false positives, amplitudes of action potentials below 20 μV were excluded to minimize the risk of mistaking artifacts or other volume-conducted activity for PSA. Analysis and interpretation of both PSA and MUAP were based on a decomposed train of five matching potentials in accordance with published standards.35–37To assess dysfunction of LMNs, PSA and discharge rates of MUAPs were determined and evaluated as described previously .21–23 To23 Increased discharge rates are caused by a loss of motor units.22 Both PSA and increased discharge rates indicate LMN dysfunction but may occur independently. Therefore, LMN dysfunction was assumed when PSA and/or discharge rates of MUAPs >20 Hz were detectable. Discharge rates of MUAPs were handled as a binary classifier (≤20 Hz/>20 Hz). The severity of PSA was graded semi-quantitatively on a four-level ordinal scale from 0 to 3 as described previously.38 The PSA was correlated with the six-level ordinal-scaled motor function of the five key muscles (C5 to T1) defined by ISNCSCI.9The PSA represent discharges of denervated or damaged  single muscle fibers.40 Needle EMGs were sampled with 10 kHz and decompositioned in a nEMG recording device . Recordings were done under acoustic control with the following settings: skin temperature >30°C, (bandpass) filter 5 Hz to 5 kHz, amplification 50 μV/Div (PSA)/0.1–5.0 mV/Div (MUAP), and sweep speed to 10 msec/Div.Based on relevant literature and broadly accepted routine practice, muscles displaying MUAP amplitudes ≥2 mV  were considered to have undergone reinnervation by collateral sprouting as a compensatory mechanism at the chronic stage nEMG.The anatomical T2-weighted MRIs were acquired on 1.5T and 3T scanners  immediately after admission to the primary care hospital and—if applicable—before spine surgery. The MRI parameters were as follows see also : slice tThe T2-weighted hyperintense MRI signal changes at the spinal cord lesion site were considered indicative of myelopathy and associated structural damage. Subsequently, the extent of myelopathy was compared with the nEMG findings  and eval41: three T2-weighted MRI axial scans per cervical segment were analyzed as a percentage of the cross-sectional area of the spinal cord. For this, the relevant sections of each cervical segment were derived from the scan closest to the cover plate, the mid-level, and the base plate of the respective vertebral body.The MRI analysis was performed by a board-certified neuroradiologist and a board-certified neurologist using conventional open-source processing software . Cervical neuroforamina were analyzed to exclude compression of nerve roots. The spinal cord segmental level (C1-T1) was assigned as a reference to the respective vertebral bodyThe area showing hyperintense signal changes representing myelopathy of the spinal cord was marked and normalized by the total cross-sectional area of the spinal cord . SubsequFinally, the extent of myelopathy for each segment was averaged over both examiners. The following formula summarizes the workflow:negative or positive. It was considered positive in case of PSA >0 and/or discharge rates of single MUAP >20 Hz were detected. For semiquantitative evaluation of PSA, a grading of 2 and 3 reflected moderate to severe LMN dysfunction, whereas a grade of 1 was considered mild LMN dysfunction. Needle EMG findings were grouped by the segmental distance from the ISNCSCI motor level. Negative numbers represent segments rostral to the lesion, whereas positive numbers stand for segments caudal to the lesion.The presence of LMN dysfunction was classified with a binary variable as either 42 matplotlib ,43 and scipy (statistics).44Data were processed, analyzed, and visualized using the Python Data Science Stack, i.e., pandas (data processing),2-test was used to test the distribution of LMN dysfunction in the above-level, at-level, and below-level grouping, the association of LMN dysfunction with high amplitudes of MUAPS and motor recovery, as well as subgroup analysis (AIS A/B versus AIS C/D).The Χ45 Categorical data are presented as median and related interquartile range (IQR) or both 25th and 75th percentiles. Exact p values are reported with α <0.05 as threshold for significance.Spearman's ρ rank correlation coefficient was used to test the association between ordinally distributed variables such as the ISNCSCI motor score or the PSA. Association was interpreted as slight (r ≥ 0.1), moderate (r ≥ 0.3), or strong (r ≥ 0.5).Within 51 months, 121 individuals met the EMSCI inclusion criteria. Of these, 78 presented with cervical SCI and were asked to participate in a pre-screening to check for eligibility. Eight individuals declined, and the remaining 70 were screened. Twenty-nine individuals were then included in the study, of whom five did not tolerate nEMG. Of the remaining 24 participants, 17  with a median age of 54 (28–59) years completed the whole study protocol including an assessment in both the acute and the chronic stage of SCI . FourteeParticipants were characterized by a relative loss of motor function in the upper extremities as follows: entire cohort (AIS A to AIS D) = 71.2%; motor complete individuals (AIS A/B) = 71.4%; motor incomplete individuals (AIS C/D) = 67.4% .n = 85) during the acute stage, two of the 17 participants declined nEMG in the chronic stage. In addition, one subject refused nEMG in three key muscles . Therefore, 72 muscles were assessed in the chronic stage.Needle EMG was performed in the acute stage 54 (42–57) days and in the chronic stage 361 (301–375) days post-injury. While all 17 participants agreed to each key muscle examination C5–T1 (n = 69) for 16 segments, with one segment missing in six individuals, two segments missing in two, and three segments missing in two additional participants. Clinical examinations (ISNCSCI) were performed at 28 (23–33) days after injury in the acute stage and 361 (301–375) days after injury in the chronic stage . Analysis of neuroforamina revealed no (apparently persistent) compression of nerve roots in any of the participants. In 10 participants, the axial scans of the MRI did not cover all relevant segments leading to missing MRI data , followed by 56.1% of the examined muscles below-level and 22.2% of the muscles above-level (Χn = 9/19) showed signs of severe PSA compared with 33% (n = 19/57) of all muscles more than one level below the motor level (n = 9/57) of muscles below-level displayed mild PSA. No acute signs of LMN dysfunction were detected in muscles more than six levels caudal to the motor level (n = 4). Only two of the nine muscles examined more than one level rostral to the motor level showed signs of LMN dysfunction. Accordingly, these findings could be confirmed by a significant negative association between PSA severity and increasing distance from the lesion site .Overall, severe LMN dysfunction, as determined by a PSA grade of 2 or 3, was found in 29 of all 85 (34%) muscles tested. Of all muscles at-level, 47% (or level . Sixteenn = 3),46 with a higher prevalence of muscles with signs of LMN dysfunction in participants with motor complete lesions . Segment-by-segment analysis revealed that the percentage of muscles with abnormal nEMG findings peaked around the lesion level and decreased over five segments caudal to the motor level . Of the 72 muscles in which nEMG was performed at both acute and chronic stages, 41 showed LMN dysfunction at the acute stage, 34 (83%) of which transitioned to display amplitudes of MUAPs ≥2 mV indicating collateral sprouting in the chronic stage . The segmental distribution of high MUAP amplitudes was relatively balanced at-level and up to six segments below —was found. Likewise, higher grades of PSA correlated moderately with the extent of myelopathy . Muscles referring to at-level myotomes displayed a slightly inferior gain in strength over time (1 motor-score-point) in case of LMN dysfunction  each between the acute and chronic stages. Muscles with signs of LMN dysfunction, however, maintained lower strength at both stages (median 1 motor-score-point) compared with muscles without signs of LMN dysfunction  and different parameters chosen for quantification of EMG results.18Surprisingly, a high prevalence of muscles with signs of LMN dysfunction (80%) was not only found in individuals with motor complete lesions (AIS A and B), where a more widespread damage because of the presumed extensive structural damage was to be expected. Even in individuals with motor incomplete SCI (AIS C/D), 51% of investigated muscles displayed LMN dysfunction. Previous studies did not provide comparable data in this respect.n = 7, 8.2%) representing clinically intact spinal cord segments at or above the motor level. Previously reported multi-segmental innervation of muscles likely explains this phenomenon, where innervation from a structurally affected segment targets a “key” muscle formally assigned to an above-level segment.47A further interesting finding was the detection of LMN dysfunction in a small number of muscles (18 and/or secondary (inflammatory response)49 underlying causes in the majority of related muscles investigated, not only close to but also remote from the lesion site. In a subset of muscles investigated (17%), only transient LMN dysfunction  was detected, which may have been caused by transient changes such as edema.50Permanent LMN dysfunction (at the acute and chronic stage) associated with the presence of structural damage detected by spine MRI suggest primary 51 Whether such a mechanism really exists and how it exactly impacts clinical outcomes is still a matter of debate.52–54Mild LMN dysfunction (grade of PSA = 1) found in 16% of muscles related to spinal segments distant from the lesion center (below-level) was associated with rather slight impairment of voluntary motor function both in the acute and chronic stages of SCI. This may have been caused by transsynaptic degeneration.55After cervical SCI, two different scenarios of neural damage can, in principle, lead to paretic muscles. In the first scenario, only the long-axon motor tracts  are disrupted. In the second scenario, both the long-axon motor tracts and the corresponding LMNs are damaged . IsolateAccordingly, it seems plausible that a combined injury of upper motoneurons and LMNs leads to more pronounced paresis in muscles with signs of LMN dysfunction. Of course, any comparison of recovery rates in muscles with and without LMN dysfunction should be treated with caution because of the ordinal nature of motor scores and potential ceiling effects.55 In case of combined long-axon motor tract disruption and LMN dysfunction, as occurs in cervical SCI, collateral sprouting of motor nerves in the peripheral nervous system—identified by high amplitudes of MUAPs indicative of reinnervation—may represent an additional compensatory mechanism for spontaneous recovery of motor function (39With respect to long-axon motor tract damage (CNS environment), several mechanisms have been discussed and identified to contribute to spontaneous motor recovery after SCI, the most prominent being collateral sprouting of uninjured axons and/or synaptic rearrangement at supraspinal and spinal levels .function .3950 and the affected spinal cord may have been compressed and distorted by dislocated spine fragments, further limiting reliable identification of the region of interest.The extent of spinal cord damage depicted by T2-weighted hyperintense MRI signal changes in cord parenchyma correlated with LMN dysfunction in respective myotomes. A more refined assessment of the ventral horn of the spinal cord gray matter, which contains the LMNs, was not feasible with reasonable confidence because of the variability of routine spine MRIs with respect to timing and imaging parameters. Moreover, the assessed MRIs were performed early after injury, where hyperintense signal changes in T2-weighted images are still vaguely defined26 Expanding on the findings of the present study, further important insights regarding force generation and fine motor skill performance in muscles with LMN dysfunction will be gained by introducing readouts from non-invasive electrical stimulation.56The present study indicates that after a cervical SCI, LMN dysfunction is extensive and muscles with signs of LMN dysfunction lag behind muscles without such signs with respect to the maximum degree of motor control up to one year after injury. These findings may help to better predict motor recovery beyond routine comprehensive SCI care. The LMN dysfunction also challenges FES-based neuroprostheses and nerve transfer surgeries.57 Even if successful axonal regrowth is achieved, the extent of motor recovery may be affected by dysfunctional LMNs.The degree of LMN dysfunction as demonstrated in this study may represent a relevant confounding factor in clinical trials aiming for axon regeneration across the injury site.


The controlled deliveryof micronutrients to soil and plants isessential to increase agricultural yields. However, this is todayachieved using fossil fuel-derived plastic carriers, posing environmentalrisks and contributing to global carbon emissions. In this work, anovel and efficient way to prepare biodegradable zinc-impregnatedcellulose acetate beads for use as controlled release fertilizersis presented. Cellulose acetate solutions in DMSO were dropped intoaqueous antisolvent solutions of different zinc salts. The dropletsunderwent phase inversion, forming solid cellulose acetate beads containingzinc, as a function of zinc salt type and concentration. Even highervalues of zinc uptake (up to 15.5%) were obtained when zinc acetatewas added to the cellulose acetate–DMSO solution, prior todropping in aqueous zinc salt antisolvent solutions. The release profilein water of the beads prepared using the different solvents was linkedto the properties of the counter-ions via the Hofmeister series. Studiesin soil showed the potential for longer release times, up to 130 daysfor zinc sulfate beads. These results, together with the efficientbead production method, demonstrate the potential of zinc-impregnatedcellulose acetate beads to replace the plastic-based controlled deliveryproducts used today, contributing to the reduction of carbon emissionsand potential environmental impacts due to the uptake of plastic inplants and animals. Controlleduptake and release of zinc from cellulose acetatemicrobeads in both water and soil for precision agriculture. Severalmaterials have been previously investigated, either as the sole componentor as parts of compositions, including calcium alginate,23 chitosan,24 carboxymethylcellulose,25 and zein.26 However, many of these biodegradable polymers have challengesblocking further adoption; for example, they are more expensive thanoil-based materials.27 Cellulose is a potentialalternative, being one of the world’s most abundant materials,accounting for 80% of biomass and is primarily derived from timberand cotton.28 The authors have recentlydemonstrated that cellulose can be used for the controlled deliveryof micronutrients to plant roots from soil.29 However, cellulose is neither easily dissolved nor easily processed,requiring derivatization to undergo dissolution using common solventsor, in the absence of a derivatization regime, the use of expensiveionic liquids to dissolve standard cellulose.30 On the other hand, a number of cellulose derivatives—suchas ethyl cellulose and cellulose acetate—are soluble in a widerange of solvents deemed “green/sustainable”, such asethanol and DMSO.31 This could allow forthe economic and sustainable deployment of cellulose-derived materialsin the field of agriculture for the controlled-release of macro- andmicronutrients.Controlling the release of nutrients canmitigate the adverse effectsof nutrient application. Depending on the nutrient format—organicor inorganic compounds—this can be achieved in a number ofways, including via the use of polymeric32 high-shear mixing,33 dropping (single channel extrusion), utilizing solvent/antisolventprecipitation by aqueous or organic solvents,34 and via electrospray.35 The choice ofantisolvent will affect various properties of cellulose-based beadsduring the precipitation/regeneration process.36 However, there is very limited literature on the preparationof cellulose acetate beads. Previous studies include the use of solutionsof cellulose acetate regenerated in an acidic antisolvent to formbeads and to induce the production of gases from which internal poresare formed.37 Cellulose acetate beads havealso been produced using high shear mixing to produce droplets, whichcan be stabilized and precipitated.38 However,beads produced in this manner have large voids in their center, whichlead to their collapse into basin-shaped particles. Base-catalyzeddeacetylation has been used to create cellulose acetate beads withlarge central voids, which have successfully been used to elute pharmaceuticals.39The production of cellulose microbeads has beendemonstrated viaa range of techniques, including membrane emulsification,40 The effects of solvent, antisolvent, and polymerconcentrations on their size and internal structure were investigatedto maximize the uptake of zinc and control its release. Porous celluloseacetate beads were produced by the addition of various zinc saltsto the antisolvent during regeneration. The uptake of zinc into thebeads was measured, with a comparative study on their release in aqueousand soil-based environments showing the possibility of controlledrelease over a long period of time.In this work, a novel and efficient methodto produce zinc-impregnatedcellulose acetate beads was developed for the controlled release ofzinc into soil. Zinc was chosen as a model micronutrient, as it isa key input in a variety of physiological processes in plants andyet is deficient in nearly half of the agricultural soils worldwide.Cellulose acetate  (9004-35-7)and zinc nitrate (228737) were obtained from Sigma-Aldrich. Zinc chloride(98%+), zinc sulfate (99%), and zinc acetate (98%) were obtained fromFisher Scientific. Sodium chloride (S9888), zinc carbonate (96466),and DMSO  were obtained from Honeywell Ltd. UltrapureDI water was produced using Veolia PURELAB Chorus (resistivity 18.2MΩ).eq S1 to determinethe maximum needle height and droplet velocity and ensure that anyparticles produced were spherical. Needle size and solution viscosityalso contribute to obtaining spherical beads, as well as determiningthe size of the droplet produced.41 Precipitatedbeads of cellulose acetate were left to settle for 24 h in the antisolventsolution, before washing with fresh antisolvent twice more for 12h each, before retrieval by filtration. Beads were dried at 50 °Cfor 48 h within a drying cabinet and were weighed periodically forthe final 24 h until no further mass loss was observed. Two solutionconcentrations of cellulose acetate (10 and 15% wt) were assessedto determine their suitability as solutions for spherical bead production.Pure celluloseacetate beads were made as follows: solutions of cellulose acetatewere prepared in volumes of 50 mL using DMSO, with dissolution initiatedwith sonication  followed by roller mixing. Droplets were produced via a needle droppingmethod using needles with internal diameter 18–30 G . Using a syringe pump, the cellulose acetatesolutions were extruded through a needle into a pool of antisolvent(DI water) below. The needle/antisolvent distance was 60 mm to ensureproduction of spherical beads. This optimal needle height was determinedexperimentally using Impregnation of zinc into cellulose acetate beads was achievedvia two different methods: (i) precipitation of cellulose acetatedroplets in zinc-containing aqueous antisolvents and (ii) additionof zinc salts into the cellulose acetate solution followed by precipitationin zinc-containing antisolvents. Using the dropping method for celluloseacetate (15% wt in DMSO), the droplets were regenerated in solutionsof zinc salts (1–12% wt zinc), followed by further rinsingin antisolvent  to remove DMSO. Thesecond method used the same antisolvent method to impregnate beadswith zinc, with the addition of zinc acetate within the celluloseacetate solution . The beads were then dried as per the previous method. Allsoaking was conducted in the appropriate zinc salt solution to achievephase inversion and zinc uptake.–1, at 25 °C (±0.1 °C).Cross-sectional imagery of the beads was obtained using X-ray computedtomography, using a Nikon XT H 225 ST CT scanner using a Tungstentarget and PerkinElmer 1620 16 bit, 2000 × 2000-pixel detector.Scans were processed using Avizo Fire (v8.0). Scanning electron micrographswere obtained using a JEOL 648 OLV scanning electron microscope. Samplepreparation involved freeze-drying and rupture to obtain access toinner portions of the beads, followed by sputter coating prior toimaging . Mechanical analysis of driedbeads was performed using an Instron 3369 at a rate of 0.1 mm min–1, using a 100 N load cell. Bead sizes were measuredusing digital callipers (RS Pro ±0.01 mm) with sample averagesobtained for n = 10–15. IR spectra were obtainedusing attenuated total reflectance FTIR (Bruker Alpha II).The viscosity of the polymersolutions was measured using a Discovery Hybrid Rheometer  using a sand-blasted 40 mm 1° cone at a gap of 50μm. Analysis was conducted by flow sweeps at shear rates between0.01 and 100 sDried beads produced using variouszinc-containing antisolvents were immersed in water, under constantstirring. Conductivity measurements were taken every 3 s using theAccumet 1330 software, allowing for end-point determination of zincrelease against the pre-prepared calibration curves. The limit ofdetection was determined to be 0.001% wt Zn, with the limit of quantificationdetermined for all zinc salts set at 0.003% wt. The quantity of water(1 L) against the quantity of beads used in each test (∼0.3g) ensured that any zinc remaining in the beads at the end point was,therefore, negligible.n = 3),soil background blank (n = 5), and spiked soil samples(n = 6) as quality assurance for the analysis , using a glass electrode asper ISO 10390:2021. This utilizes solutions of barium sulfate, magnesiumsulfate, and sulfuric acid to determine the ionic strength of thesoil, combined with pH measurements of extracts. The samples wereprepared in triplicate. The beads were dried at 50 °C for 48h within a drying cabinet and were weighed periodically for the final24 h until no further mass loss was observed. The release of zincfrom the beads into a soil matrix was performed using soil samplesmaintained at 25% hydration. Soil cuvettes were 3D-printed using aStratasys F 270 FDM unit using PLA filament 1i–iii. Tanalysis 1iv,v.Using 15%wt cellulose acetate solution in DMSO, beads were produced via thedropping method and characterized. Optical imagery shows sphericalwhite beads with a smooth surface 2A. Inves18 It was observed that antisolvent density had a profound effect onthe ability to produce spherical beads. An increase in the antisolventdensity above 1.01 g/mL required a lower distance between the needleand the antisolvent to ensure bead sphericity. These distances wereused to determine the droplet velocity at impact, which was envisagedto exert an influence on the sphericity of the droplets once theyenter the antisolvent, due to the increasing energy of the impactat higher speeds. Higher needle-to-antisolvent distance deformationsoccurred on impact, resulting in disc- or pancake-like beads  were examinedto ascertain if this trend was applicable to zinc species with differentcounter-anions. These were chosen to ensure that they met environmentalregulations and were generally regarded as safe (GRAS) by the Foodand Drug Administration,42 with no significantconcerns for human exposure, environmental fate, ecological effects,or effects on animals grazing in areas with zinc salts applied.43 Zinc products have been approved for use aspesticides, herbicides, and fungicides by US state agencies.44 This is primarily due to the binding potentialof these zinc compounds in soil, which is dependent on soil texture,pH, organic carbon content, and soil cation exchange capacity.45 Zinc salts  are added to irrigationwater to increase the micronutrient content of soils.46In a previouswork by the authors,cellulose beads were first formed by precipitation in water, dried,and then immersed in a zinc solution to impregnate them with the metal.The capacity for cellulose acetate beads to uptakezinc was initially tested across the five zinc salts at various concentrationsof 1, 2, and 5% wt of zinc [Zn], with significant variations observedacross the different zinc species 4.Figure S1). Thismade the nitrate salt be discounted as a compound of interest. Zincchloride was tested because of its high solubility in water; however,upon testing zinc chloride concentrations above 5% wt [Zn], the beadswere found to be prone to hydrolysis during drying. This resultedin beads decomposing during the drying phase . Aqueous zinc chloride is a known solvent for bothcellulose and cellulose acetate and acts as a mediator for acid hydrolysis,which reduces the effectiveness of this compound at higher concentrationsdespite promising initial results.48 Zinc acetatewas found to have high solubility in water; however, solutions ofzinc acetate rapidly become too dense to support the production ofspherical beads: the impact of the droplets on the surface of thezinc acetate antisolvent solutions at concentrations greater than5% wt [Zn] resulted in oblong spheroid shapes rather than sphericalbeads .51 The increase in zinc uptake as the anion chargedensity decreases in magnitude suggests that an increased charge couldinhibit the diffusion of the anion into the particles at the pointof phase inversion of the droplet of cellulose acetate solution, orduring later stages of soaking. The increasing energy required toremove anions of higher magnitude from hydration could work in parallelwith the preference for salts to form interactions with differentmoieties, as observed for anion uptake in other systems.51 Overall, the direct impact of the counter anionappears to determine the quantity of zinc that is taken up by thebeads during regeneration which, in turn, affects the total zinc availablefor release, and the rate at which it egresses from the beads.The order of increased uptake of these anions wasfound to followthe lyotropic and Hofmeister series,e anions 5. The tr2 antisolvent solution (5% wt Zn). Theseproduced beads with zinc content of 11.7 and 15.5%, respectively,higher than the values obtained for the ZnOAc2 antisolventalone  with zinc acetate (1 and 2% wt) in DMSO were produced , with droplets of these precipitatedin aqueous ZnOAcone cfr. 1.Figure S3), which allowed discounting bead diameteras a potential source of variation for total zinc-uptake during precipitation,nor for the variation in zinc release in water (Table S1). Using the data in Table S1, comparing the retention times of beads precipitated in 1 and 5%wt zinc salts shows that the beads with a higher concentration tendedto elute their zinc over a slightly longer time period (between 10and 12% longer on average between 1 and 5% wt). Release profiles forzinc salts across all beads in water showed a range of 47 to 67 minrelease time (Table S1).The release of zinc fromthe beads in distilled water shows that the rate of release is dependenton both the exact zinc compound used, and the concentration of thezinc within the beads 6. No sig54 This is a phenomenon that iswitnessed when charged ions within delivery substrates are exposedto aqueous surroundings, causing a rapid migration from the substrate.55 The comparison between 1% wt zinc sulfate andzinc acetate shows that although both antisolvents can impregnatesimilar amounts of zinc within the beads (Table S1). This difference in retention time suggests that the associatedanions impart a physical or chemical modification, which inhibits,or induces, the release of zinc. This is perhaps a result of the differencein charge densities between the SO42– and OAc– anions  and20% (5% wt [Zn] Zn(OAc)2 w/2% [Zn] in polymer solution)for these beads 2) have a ∼16 days time span before fullrelease of all zinc  andonto hydroxides present in the soil, existing in equilibrium betweenthese solids and the water present in the interstitial voids withinthe soil.59 Soil–water partitioncoefficients for zinc species show that zinc ion solubility increasesby a factor of 5 with every pH unit decrease. These data imply thatthere is an advantage for zinc sulfate with regard to diffusion fromcellulose acetate beads to the immediate aqueous environment due toits pKa value, which in turn suggests that sulfate-based zinc compoundswill undergo diffusion and transfer from within bead to the immediatesoil–water matrix at a rate that is greater than that for thezinc acetate beads. As such, higher pKa values could potentially ensurea longer release time due to the lower dissociation constant for thesaid salts. It could also be posited that the overall solubilitiesof the various zinc compounds in water affect these results: the solubilityin water for the acetate dihydrate variant (43 g/100 mL) is lowerthan that for the sulfate heptahydrate variant (54 g/100 mL).61 This increase in solubility could explain the increased speed atwhich the zinc sulfate entered the surrounding moist soil, aided bythe osmotic pressure.The release from the 12 and 5% wt [Zn] zinc sulfatesamples 7Ahas a 47 showing that the presenceof zinc initiates an acid hydrolysis reaction at room temperature,degrading the glycosidic bond and producing glucose.47 In such a case, it could be expected that the hostile conditionscreated by the presence of zinc chloride (or other zinc compounds)within the bead could expedite the physical deterioration of the beadchemically or encourage biological degradation through the productionof sugars. Additionally, oligomers produced during the bead decompositionmight enhance the soil cation exchange capacity, which is a highlydesirable feature capable of improving the retention of other nutrients.Furthermore, such oligomers, likely negatively charged, may also preventphosphorus strong adsorption on iron and aluminum oxides. These twohypotheses shall be tested in further studies. Other factors affectingZn2+ release from the cellulose substrate include localsalinity and pH, as other lignocellulosic materials have shown tohave preference for retention of other bivalent metal ions under specificsalinity and alkaline conditions.62A direct comparison with other controlledrelease fertilizers ismade complex not only from the wide range of supports and materialsbut also from the large number of factors that can affect release,including the rates of biological and chemical degradation of thebeads, the form of zinc salt present, and the presence of water inthe form of humidity. For example, zinc compounds such as zinc chlorideand nitrate are known to enhance the chemical breakdown of celluloseand its derivates,63 a shorter timespan than that reported in this study. The releaseof zinc sulfate from beads produced using microcrystalline celluloseoccurred within ∼400 min within both loamy and sandy soils.29 Other formats involve coating urea with Zn-fortifiednano-bentonite and ZnO nanoparticles using various binders which couldrelease zinc, but with full zinc release occurring at around 30 days.64To date, this is the first example within the literature of a controlledrelease fertilizer produced using cellulose acetate as the sole matrixcomponent. Comparing this material against other composite materialsas controlled release fertilizers is favorable toward the celluloseacetate bead method: zinc sulfate encapsulated within manganese hollow-coreshells was released within ∼33 days,Figure S4), with no fracturing occurringunder loading. Previous works on the mechanical strength of cellulosemicrobeads found that these also exhibited no fracture at compression,with 59 N required to achieve compression at 30%.32Compressiontesting carried outon the cellulose acetate beads of diameters ranging from 1.75 to 1.93mm required ∼10 N to achieve approximately 30% compression(In this study, a novelmethod with potential for scale-up to producezinc-impregnated cellulose acetate beads was developed using zinc-containingantisolvents to induce uptake during regeneration. Increased levelsof encapsulated zinc compounds were achieved by utilizing zinc saltsthat possessed counter-anions with lower magnitudes of radial chargedensities, by using different concentrations of cellulose acetateand by impregnating cellulose acetate solutions with zinc acetateprior to precipitating the droplets of this solution in zinc-containingantisolvents. Both the quantity of zinc within these beads and theirrelease time in aqueous environments was found to depend highly onthe form of the zinc salts present, with this release monitored byconductivity measurements to track the concentration of the zinc released.The level of zinc impregnation achieved surpass that of cellulosebeads reported in the literature, with release times from the beadsexceeding recent zinc delivery methods. Release tests in soil showedslow delivery over time for both zinc sulfate and zinc acetate beads,with the potential to deliver zinc ions to soil over timescales exceeding45 days. These results show that the controlled release of micronutrientscan be achieved using a biodegradable carrier, potentially eliminatingthe need to use the fossil-fuel-derived plastics currently used inagriculture.


BMI) in the Electronic Medical Records and Genomics (eMERGE) network . Analyses were conducted in two ancestry groups (European and African) and three age ranges . For PRSBMI calculations, we evaluated five LD reference panels and three sets of GWAS summary statistics of varying sample size and ancestry. PRSBMI performance increased for both African and European ancestry individuals using cross-ancestry GWAS summary statistics compared to European-only summary statistics . The effects of LD reference panels were more pronounced in African ancestry study datasets. PRSBMI performance degraded in children; R2 was less than half of teenagers or adults. The effect of GWAS summary statistics sample size was small when modeled with the other factors. Additionally, the potential of using a PRS generated for one trait to predict risk for comorbid diseases is not well understood especially in the context of cross-ancestry analyses – we explored clinical comorbidities from the electronic health record associated with PRSBMI and identified significant associations with type 2 diabetes and coronary atherosclerosis. In summary, this study quantifies the effects that ancestry, GWAS summary statistic sample size, and LD reference panel have on PRS performance, especially in cross-ancestry and age-specific analyses.Polygenic risk scores (PRS) have led to enthusiasm for precision medicine. However, it is well documented that PRS do not generalize across groups differing in ancestry or sample characteristics e.g., age. Quantifying performance of PRS across different groups of study participants, using genome-wide association study (GWAS) summary statistics from multiple ancestry groups and sample sizes, and using different linkage disequilibrium (LD) reference panels may clarify which factors are limiting PRS transferability. To evaluate these factors in the PRS generation process, we generated body mass index (BMI) PRS (PRS Polygenic risk scores (PRS) provide individualized genetic estimates of a phenotype by aggregating genetic effects across hundreds or thousands of loci, typically from genome-wide association studies (GWAS). PRS are potentially a powerful source of increased prediction performance, even when combined with family history ,2. HowevSeveral outstanding questions surrounding PRS, especially within the context of adiposity traits and BMI, warrant further investigation. For instance, when cross-ancestry summary statistics  are available, can they be used to improve prediction performance in individuals from one or more different ancestry groups? We need a more thorough evaluation of the potential prediction performance gain (or loss) in African ancestry individuals when cross-ancestry GWAS summary statistics are used to estimate the SNP weights. In addition, we need to improve our understanding of the impact of the composition of the linkage disequilibrium (LD) reference panel in combination with cross-ancestry GWAS summary statistics on PRS prediction performance. For prediction of BMI specifically, how does prediction performance differ for individuals in different age groups, especially those who are not adults ? Additionally, how much these different variables impact the PRS performance when considered together is important to explore. Developing a deeper understanding of which features  have the greatest impact of PRS performance will help the field develop future studies and strategies around clinical risk prediction with PRS. The degree to which increased GWAS sample size increases prediction performance regardless of these other factors is also important to determine. Finally, there is potential for using a PRS generated for one trait to predict risk for comorbid traits. Understanding how much the different elements of PRS generation affects associations with clinical comorbidities of obesity is of great importance for precision medicine.BMI) across 90 different combinations of analyses . Investigation of these variables elucidates our understanding of the factors that affect PRS performance and transferability across ancestries and populations, especially within the context of BMI, as well as the potential of using PRSBMI to predict risk for comorbid disease.We comprehensively investigated the influence of these factors on the performance of PRS using the Electronic Medical Records and Genomics (eMERGE) Network dataset. eMERGE is an NIH funded consortium that combines participants from multiple electronic health record (EHR) linked biobanks . In the BMI for all individuals within each combination where the following elements of the model varied: 1) LD panels that differed in ancestry, 2) GWAS summary statistics with variable ancestry composition, and 3) GWAS summary statistics for two different sample sizes. The details for each of these are provided more below. The data was also split by ancestry and age group, and we statistically compared PRSBMI performance between all the different groups – in total, 90 sets of PRSBMI were calculated separately and then compared. We first estimated the effect and significance of each variable  on PRS performance. Next, we estimated how much each variable affects PRSBMI performance when all are modeled together, and finally we analyzed the potential clinical associations by testing the PRSBMI for association with common comorbid conditions from the EHR. For the primary results related to LD panel or ancestry of summary statistics and test data, we restricted analyses to adults as the other age groups were limited in sample size. In the following sections, we describe all these elements in more detail.The electronic Medical Records and Genomics (eMERGE) network dataset is an NIH funded consortium that combines participants from multiple electronic health record (EHR) linked biobanks. In this study, we included 75,661 individuals with available genetic and phenotypic data. The individuals in the eMERGE dataset include multiple ancestry groups – genetically inferred ancestry was assigned by the eMERGE consortium  – and a We obtained published GWAS summary statistics from the GIANT consortium  to use and degree relatives or higher), and within the White British ancestry subset   included as covariates. BMI, defined as weight in kilograms divided by squared height in meters, was first inverse-rank normal transformed.For the second set of summary statistics, we performed a GWAS in the individuals of European ancestry from the UK Biobank (UKBB). Individuals were first filtered by low quality samples , relatedness (no 2ncestry) ; a totalncestry) ) > 0.30,After calculation of BMI GWAS summary statistics in each of the two datasets of UKBB individuals of European ancestry, we harmonized variants across all datasets used . For the remainder of downstream analyses, we kept only those variants that were present in all datasets, and additionally excluded any strand-ambiguous SNPs , and retained only biallelic variants; in total, 2,014,457 variants were retained for analyses.BMI calculations: 1) all of 1000 Genomes (1KGAll) , 2) 1000 Genomes European ancestry (1KGEUR) (N=503), 3) 5,000 randomly selected European ancestry individuals from the UK Biobank (UKBBEUR), 4) 5,000 randomly selected individuals from all of UK Biobank (UKBBAll), and 5) up to 5,000 randomly selected individuals from the dataset for which PRSBMI were being calculated for in the eMERGE dataset (referred to as test data henceforward). These panels were chosen to test for differences in ancestry distribution and sample size on PRS performance.Five different LD reference panels were used for each set of PRSBMI were calculated using pruning and thresholding method via PRSice v2.1.9 .For each comparison set, PRSe v2.1.9 . We chos2 for PRSBMI was calculated by subtracting the R2 using a model with only the covariates from the R2 of the model using the covariates and the PRSBMI (the default option in PRSice). Statistical differences between model performances from different iterations were determined using the Wilcoxon rank-sum test to compare the distributions of the squared residuals generated from the model for all individuals in the iteration; for comparisons between the same set of individuals, the paired Wilcoxon rank-sum test was used. When testing which of the five LD panels performed the best, we used a Bonferroni-corrected threshold of 0.05/10 = 0.005 (ten comparisons between five LD panels). When comparing the best performing PRSBMI across ancestries and summary statistics using their best LD panel, we used a Bonferroni threshold of 0.05/25 = 0.002 (25 comparisons between the five LD panels used).Incremental RWe modeled all evaluated features together in the following linear regression model:Sumstat subscript is defined as a set of GWAS summary statistics, and the Test subscript is defined as a set of test individuals that PRS prediction is being assessed in. We quantified the variance in R2 that could be explained by each of these different variables using type II sum of squares from ANOVA. The sum of squares of variables involving ancestry were summed together; an interaction term between summary statistics ancestry and test data ancestry was included to identify whether the ancestry of summary statistics and test data matched.Where the BMI. For each Phecode, individuals were classified as a case for the condition if there was at least one occurrence of the respective Phecode in their EHR record; individuals were classified as a control for that condition if there was no occurrence of the Phecode. This classification is a rule-of-one instance of a Phecode to define case status. For each eMERGE ancestry subgroup, we selected the best performing PRSBMI i.e., the PRSBMI with the highest R2, and tested the association of the PRSBMI with these ten clinical conditions using a logistic regression model. PRSBMI was first mean-centered and standard deviation was set to 1. Sex, age, age2, and the first five genetic PCs were included as covariates.We selected the ten most frequent Phecodes  from theThe ‘ggplot2’ R package was used for plotting, with the ‘geom_signif’ package used to include significance bars. The association results were plotted using PheWAS-View .BMI performance compared to European ancestry LD panels  had an R2 of 0.0109 (p = 4.94×10−20). When using the GIANT summary statistics, the top PRSBMI R2 was 0.0149 using 1KGAll as the reference panel. The PRSBMI calculated using the best European ancestry panel (1KGEUR) resulted in a R2 of 0.0141, but this difference between these two reference panels was not Bonferroni significant (p = 0.037). However, the 1KGall LD panel performed significantly better than the two UKBB LD panels . The test data LD panel performed the second-best with an R2 of 0.0142, and significantly outperformed the UKBB European LD panel (p = 4.78×10−5). For adults of European ancestry, we observed more significant differences in performance when using the GIANT summary statistics compared to the down-sampled UKBB summary statistics. The 1KGAll LD panel performed the best with a R2 of 0.0612. It also significantly outperformed all other LD panels . We note that this increase was larger when using the GIANT summary statistics but was still present when using the UKBB summary statistics. When using the UKBB summary statistics, the choice of LD panel had a much smaller impact on prediction performance. While the 1KGAll LD panel performed the best, the difference in performance was much less significant between the next best performing LD panel . The difference between the best and worst performing scores – LD panel using 1KG all versus 1KG European – was also much less significant (p= 1.15×10−12). These results suggest that the choice of LD panel particularly matters when calculating PRSBMI using cross-ancestry GWAS, or for African ancestry individuals when the GWAS summary statistics are derived from European ancestry individuals.For adults of African ancestry, when using the down-sampled UKBB GWAS summary statistics, using either cross-ancestry or African ancestry test data LD panels significantly improved PRSD panels . When usEUR LD panel performing best . The 1KGAll LD panel was the worst performing LD panel with an R2 of 0.0185, and this difference between the 1KGEUR LD panel was significant after multiple hypothesis correction (p = 5.08×10−7).However, we did observe a slight decrease in the impact of the choice of LD panel when using the full UKBB summary statistics for adults; again, the largest differences were observed in adults of African ancestry, but differences in performance across LD panels were not as significant. The test LD panel performed second best with the 1KG2 values of the PRSBMI were significantly higher when calculated for European ancestry adults than adults of African ancestry, even when using the cross-ancestry GIANT summary statistics .As expected, the Ratistics . When us2 when using the GIANT summary statistics was higher than the R2 when using the down-sampled UKBB summary statistics with their respective best LD panel (GIANT (1KGAll LD panel): R2 = 0.0149, UKBB (test data LD panel): R2 = 0.0140; p = 0.038). This difference was not statistically significant after multiple hypothesis correction. However, the GIANT summary statistics with the 1KGAll LD panel did significantly outperform the UKBB summary statistics with all other LD panels. When keeping the LD panel constant, the PRSBMI calculated using the GIANT summary statistics resulted in higher R2 than using the UKBB summary statistics for all LD panels except for the test data LD panel, and this difference was statistically significant for the 1KGAll (p = 1.55×10−33), 1KGEUR (p = 6.78×10−18), and UKBBAll (p = 1.28×10−15) LD panels. Somewhat surprisingly, we observed higher R2 values for European ancestry adults when using the cross-ancestry GIANT summary statistics versus the down-sampled European UKBB summary statistics (R2GIANT = 0.0612 versus R2UKBB = 0.0590), with this difference being statistically significant (p = 6.26×10−4); the best performing LD panel for both set of summary statistics was 1KGAll.In African ancestry adults, the R2.We also compared prediction performance in all individuals using the full  European UKBB GWAS versus the European UKBB GWAS down-sampled to GIANT’s sample size  . For con2 values for adults and teenagers, with substantially reduced performance in children . Somewhat surprisingly, teenagers consistently had higher R2 than adults across all analyses, although these differences were much less significant than those compared with children.Across different ancestries and summary statistics, we broadly observed similar Rchildren . R2 valuBMI performance when considered together , while the number of individuals used to calculate the GWAS summary statistics only accounted for 1.9% of variance explained in PRSBMI R2. Per previous sections, while number of individuals used for summary statistics resulted in significant differences in PRSBMI performance, its overall impact when modeled jointly with all the other factors in the context of these analyses seemed to be small.While we observed significant differences due to ancestry, age, and number of individuals used to calculate summary statistics, we aimed to quantify the effect of these different variables on PRStogether . We obseBMI was associated with clinical comorbidities, we performed a Phenome-Wide Association Study for ten clinical conditions (BMI was tested for association with diagnosis codes (Phecodes) to evaluate whether the polygenic background for BMI associates with these clinical diagnoses. The PRSBMI was significantly associated with several of the most frequent Phecodes in eMERGE, particularly in European adults ; this was a positive control. In European ancestry individuals, the best performing PRSBMI was also significantly positively associated with type 2 diabetes (pEUR = 1.04×10−102), essential hypertension (pEUR = 7.12×10−56), coronary atherosclerosis (pEUR = 3.61×10−26), hyperlipidemia (pEUR = 4.38×10−16), depression (pEUR = 1.95×10−13), hypercholesteremia (pEUR = 3.64×10−15), asthma (pEUR = 3.13×10−13), and diverticulosis (pEUR = 0.0017). These associations were less statistically significant in African ancestry individuals, which had much lower sample size, and many associations were no longer significant after Bonferroni correction. Only type 2 diabetes (pAFR = 1.2×10−5) and coronary atherosclerosis (pAFR = 0.001) were significantly associated with the PRSBMI in African ancestry adults. We also looked at the prevalence of each condition per PRS quintile for the most significantly associated conditions . The much smaller sample sizes of the Phecodes in these age groups may have also contributed to the lack of statistically significant results – most of these diagnoses are adult-onset conditions.To determine whether the PRSnditions . Here, tn adults . As expenditions . The casSomewhat unintuitively, African ancestry LD panels performed best for African ancestry individuals, regardless of whether European ancestry or cross-ancestry GWAS summary statistics were used. We observed minimal impact of the choice of LD panel when both test data and summary statistics were of European ancestry. These results suggest that as long as either the test data or GWAS summary statistics are of similar ancestry, or the test data and LD panel are of similar ancestry, the difference in PRS performance may be minimal as compared to if all the GWAS summary statistics, test data, and LD panel are all of the same ancestry. We also observed significantly decreased PRS performance in children compared to adults and teens, with the GWAS used in this study being conducted on adult populations.While the findings in this study highlight many important strategies for performing PRS in different ancestry and age groups, there are limitations that should be addressed in future studies.BMI performance. Finally, repeating these types of analyses with different PRS methods would be useful as novel PRS methods are being developed on a regular basis, many of which incorporate ancestry in different ways.First, inclusion of analyses that evaluate how different proportions of non-European ancestry individuals affect the prediction performance of PRS would be useful. The GIANT summary statistics we used in this study are only about 6% non-European ancestry. It may be useful to see how the PRS prediction performance changes in both non-European and European ancestry datasets as a function of the proportion of non-European ancestry samples included in the GWAS. Such analyses may be possible by combining African ancestry individuals from these different datasets. These analyses will be possible once larger datasets that include non-European ancestry cohorts are publicly available or could be tested by analyzing other traits with larger African ancestry GWAS. Future analyses could also include sex-stratified GWAS and comparison sets to evaluate the influence of sex on PRSOverall, this study demonstrates the importance of expanding non-European ancestry data resources for PRS, specifically in the generation of GWAS summary statistics and LD reference panels. Failure to do so reduces the impact of PRS in diverse populations and increases the potential for continued health disparities, especially in precision medicine where genetics is being integrated into clinical care.Supplementary-9789811270611_0040