The phase 3 VELIA trial evaluated veliparib with carboplatin/paclitaxel and as maintenance in patients with high-grade serous ovarian carcinoma.Patients with previously untreated stage III–IV high-grade serous ovarian carcinoma were randomized 1:1:1 to control (placebo with carboplatin/paclitaxel and placebo maintenance), veliparib-combination-only (veliparib with carboplatin/paclitaxel and placebo maintenance), or veliparib-throughout (veliparib with carboplatin/paclitaxel and veliparib maintenance). Randomization stratification factors included geographic region (Japan versus North America or rest of the world). Primary end point was investigator-assessed median progression-free survival. Efficacy, safety, and pharmacokinetics were evaluated in a subgroup of Japanese patients.n = 23), veliparib-combination-only (n = 30), and veliparib-throughout (n = 25) arms. In the Japanese subgroup, median progression-free survival for veliparib-throughout versus control was 27.4 and 19.1 months . In the veliparib-throughout arm, grade 3/4 leukopenia, neutropenia, and thrombocytopenia rates were higher for Japanese (32%/88%/32%) versus non-Japanese (17%/56%/28%) patients. Grade 3/4 anemia rates were higher in non-Japanese (65%) versus Japanese (48%) patients. Early introduction of olanzapine during veliparib monotherapy maintenance phase may help prevent premature discontinuation of veliparib, via its potent antiemetic efficacy.Seventy-eight Japanese patients were randomized to control (Median progression-free survival was numerically longer in Japanese patients in the veliparib-throughout versus control arm, consistent with results in the overall study population. Pharmacokinetics were comparable between Japanese and non-Japanese patients. Data for the subgroup of Japanese patients were not powered to show statistical significance but to guide further investigation. Ovarian cancer is diagnosed in an estimated 314,000 women worldwide annually accounting for approximately 207,000 deaths ). This was comparable with the overall study intention-to-treat population, in which median progression-free survival was 23.5 months in the veliparib-throughout arm compared with 17.3 months in the control arm  . In. In27]. Fig. Plasma concentrations of veliparib in Japanese and non-Japanese patients are shown in In both Japanese and non-Japanese subgroups, the median number of cycles of placebo/veliparib received was higher in the control arm compared with the veliparib-throughout arm . The median number of cycles of carboplatin was six for both arms in both subgroups , and the median number of cycles of paclitaxel was six for both arms in both subgroups .An overview of treatment-emergent adverse events is presented in Table Common any grade adverse events with ≥ 10% higher rate in the veliparib-throughout versus control arm were nausea and thrombocytopenia (Japan and non-Japan subgroups), and anemia and vomiting (non-Japan subgroup only). Rates of alopecia and of peripheral sensory neuropathy were ≥ 10% higher in the control arm versus the veliparib-throughout arm in Japan subgroup only.In the veliparib-throughout arm, rates of any grade alopecia, nausea, neutropenia, and thrombocytopenia were > 10% higher in the Japanese subgroup compared with non-Japanese subgroup. Rates of grade 3/4 leukopenia and neutropenia were > 10% higher in the veliparib-throughout arm in the Japanese subgroup compared with the non-Japanese subgroup. Conversely, rates of any grade and grade 3/4 anemia were > 10% higher in the veliparib-throughout arm in the non-Japanese subgroup compared with the Japanese subgroup.n = 5, 20%) and the non-Japanese  subgroups. Nausea most frequently led to discontinuation among patients in the Japanese subgroup in the veliparib-throughout arm . The rate of treatment-emergent adverse events leading to veliparib dose interruption and/or reduction was comparable for the veliparib-throughout arm in the Japanese  and the non-Japanese  subgroups. The most frequently reported treatment-emergent adverse events leading to veliparib interruption/reduction (≥ 20% of patients) for patients receiving veliparib-throughout in the Japanese subgroup were nausea , neutropenia , and thrombocytopenia .The rate of treatment-emergent adverse events leading to veliparib discontinuation were comparable for patients in the veliparib-throughout arm in the Japanese  than the combination therapy phase  in the Japanese subgroup.Rates of gastrointestinal and hematologic adverse events of special interest by treatment phase are described in Table n = 2, combination with prochlorperazine or granisetron: n = 2). None of the six patients receiving olanzapine on the first day of veliparib maintenance therapy prematurely discontinued veliparib due to nausea and/or vomiting.For nausea and/or vomiting, antiemetics were used to prevent premature discontinuation of veliparib or to maintain quality of life. At the time of the interim database lock, metoclopramide (68%), olanzapine (56%), prochlorperazine 44%), and granisetron (40%) were frequently administered during veliparib monotherapy maintenance phase for Japanese patients in the veliparib-throughout arm  in the veliparib-throughout arm and six patients (1.7%) in the control arm had a treatment-emergent adverse event leading to death; none were considered related to veliparib/placebo by the investigator.In the subgroup analysis presented here, Japanese patients with high-grade serous ovarian carcinoma who received veliparib combined with platinum-based chemotherapy and continued as maintenance experienced numerically longer progression-free survival compared with Japanese patients who received platinum-based chemotherapy alone. This was consistent with the previously published primary analysis of this trial . It is nBRCA status was required for randomization, it was a significant problem for patients to await initiation of chemotherapy for approximately 2 weeks until gBRCA results were available. Provisional pathologic diagnosis using frozen tumor sections takes only several hours from biopsy to diagnosis, with a high concordance rate between rapid and definitive diagnoses [BRCA blood sampling allows definitive diagnosis and gBRCA testing to be performed in parallel, enabling earlier initiation of study treatment.As giagnoses , 29. RapP value < 0.0001 [The most common adverse events observed in Japanese and non-Japanese patients in the veliparib-throughout arm were nausea  and neutropenia (100% and 74%), and these adverse events occurred at greater frequencies than in the respective control arms . The toxicity profile observed in the veliparib-throughout arm for the Japanese subgroup is comparable with that observed in small phase 1 studies of veliparib as a single agent or combined with carboplatin/paclitaxel in patients with ovarian cancer or other solid tumors , 26. The< 0.0001 . DiffereAlthough pharmacokinetic drug profiles are known to differ among patients of different ethnicities , the resBRCA mutations is numerically higher among Japanese vs non-Japanese subgroups. Some reports have indicated increased hematologic toxicity in BRCA mutation carriers; however, a recently reported subanalysis of the VELIA trial showed that gBRCA status did not impact safety in this study [With the comparable veliparib pharmacokinetic profiles in Japanese and non-Japanese patients, the reason for observed differences in frequency of some adverse events between the populations is unclear. Different polymorphisms between populations may lead to differential sensitivity to drug. In a previously reported study, it was hypothesized that the differences in allelic distribution in genes involved in paclitaxel metabolism or DNA repair between Japanese and Western populations may result in differential sensitivity to paclitaxel. However, no significant associations were identified between adverse event frequency and the polymorphisms explored . In the is study . AdditioThe present study assesses the efficacy and safety of PARP inhibitor therapy during combination chemotherapy and continued as maintenance in patients with newly diagnosed ovarian cancer. Other studies in newly diagnosed patients have generally evaluated PARP inhibitors as maintenance therapy after combination chemotherapy only , 36, 37.Although the analysis of progression-free survival in Japan subgroup was prespecified, it was not powered for statistical significance and is limited by the relatively small number of patients. While baseline demographic and disease characteristics were generally balanced between treatment arms within the larger non-Japanese subgroup, there were some imbalances within the Japanese subgroup.Conclusions drawn on differences in rates of specific adverse event rates between Japanese and non-Japanese subgroups are limited by the relatively small number of patients in the Japanese subgroup. Despite the small numbers in the Japanese subgroup, there are consistencies between the observations in this study and in other previous reports. For example, the higher rate of neutropenia observed in the Japanese subgroup vs the non-Japanese subgroup is consistent with a previous report indicating higher rates of grade 3/4 neutropenia for Japanese vs American patients with non-small cell lung cancer receiving carboplatin and paclitaxel .n = 14, 64%; combination: n = 21, 84%) and non-Japanese  subgroups in the veliparib-throughout arm. PARP inhibitors including olaparib, niraparib, and rucaparib are categorized as moderate to high emetic risk [Although nausea was common, it was predominantly low-grade. Nausea was also reported less frequently during the maintenance phase than the combination phase in both Japanese (maintenance: tic risk . With sutic risk . ConsideVeliparib combined with chemotherapy followed by veliparib maintenance therapy provided numerically longer progression-free survival compared with chemotherapy alone, with a generally manageable safety profile in Japanese patients with high-grade serous ovarian carcinoma. These data support the efficacy and safety of veliparib combined with platinum-based chemotherapy and continued as maintenance in this population at the dose administered to the global population.


There are a paucity of randomised data on the optimal timing of invasive coronary angiography (ICA) in higher-risk patients with non-ST elevation myocardial infarction (N-STEMI). International guideline recommendations for early ICA are primarily based on retrospective subgroup analyses of neutral trials.The RAPID N-STEMI trial aims to determine whether very early percutaneous revascularisation improves clinical outcomes as compared with a standard of care strategy in higher-risk N-STEMI patients.RAPID N-STEMI is a prospective, multicentre, open-label, randomised-controlled, pragmatic strategy trial. Higher-risk N-STEMI patients, as defined by Global Registry of Acute Coronary Events 2.0 score ≥118, or >90 with at least one additional high-risk feature, were randomised to either: very early ICA±revascularisation or standard of care timing of ICA±revascularisation. The primary outcome is the proportion of participants with at least one of the following events  at 12 months. Key secondary outcomes include major bleeding and stroke. A hypothesis generating cardiac magnetic resonance (CMR) substudy will provide mechanistic data on infarct size, myocardial salvage and residual ischaemia post percutaneous coronary intervention. On 7 April 2021, the sponsor discontinued enrolment due to the impact of the COVID-19 pandemic and lower than expected event rates. 425 patients were enrolled, and 61 patients underwent CMR.The trial has been reviewed and approved by the East of England Cambridge East Research Ethics Committee (18/EE/0222). The study results will be submitted for publication within 6 months of completion.NCT03707314; Pre-results. This randomised trial sought to test whether a very early invasive strategy in higher-risk non-ST elevation myocardial infarction patients improves clinical outcomes compared with standard care. An early invasive strategy in this group is recommended in international guidelines, but is as yet unsupported by the primary outcome of an appropriately sized randomised trial.Randomised controlled pragmatic strategy design.A cardiac MR substudy will provide mechanistic data on infarct size, myocardial salvage and residual ischaemia post percutaneous coronary intervention.Due to the effects of the COVID-19 pandemic on clinical services and a lower than expected primary outcome event rate, trial recruitment was terminated early after enrolment of 425 patients (18.4% of intended).Historical data indicate that an invasive strategy during index hospital admission in non-ST elevation myocardial infarction (N-STEMI) improves composite ischaemic outcomes, with maximal benefit seen in those at highest baseline risk for future major adverse cardiovascular events (MACE).A series of randomised trials have investigated this concept in unselected N-STEMI populations, thereby testing whether early revascularisation (<24 hours) improves clinical outcomes as compared with delayed or standard of care approaches.19The application of the Global Registry of Acute Coronary Events (GRACE) score in prior trial subgroup analyses has potential for risk stratification of those patients that may benefit most from expeditious revascularisation . A GRACE9Given that currently available data are inconsistent and of insufficient scientific quality to inform best practice, a contemporary trial that prospectively investigates the timing of revascularisation in GRACE score defined high-risk N-STEMI is required to confirm or refute these prior observations.The RAPID N-STEMI trial enrolled patients across 32 hospitals with on-site cardiac catheter laboratories in the UK. Potential participants who experienced symptoms within 12 hours prior to admission were assessed on attendance to hospital and the research team alerted if a diagnosis of N-STEMI was suspected. N-STEMI was defined as: (1) the presence of cardiovascular symptoms suggestive of myocardial ischaemia and (2) elevation in high-sensitivity troponin (hs-Tn) I or T. Risk stratification using the GRACE 2.0 score was then performed. Patients in whom the GRACE 2.0 score was ≥118, or ≥90 with at least one additional feature of high-risk presentation were deemed as higher risk. The full inclusion and exclusion criteria are listed in Patients were enrolled after obtaining verbal consent once eligibility was confirmed in the emergency department or appropriate receiving unit. Participants were then randomised in a 1:1 fashion to either: group (A) very early ICA with a view to revascularisation or group (B) standard of care timing of ICA with a view to revascularisation. Research team members had 6 hours from hospital admission to randomise verbally consented patients who met all eligibility criteria .http://www.sealedenvelope.com. Those assigned to very early angiography were transferred to the cardiac catheter laboratory as soon as possible. Teams were encouraged, but not mandated to achieve a randomisation to vascular sheath insertion time of less than 90 min. Timing of standard of care ICA was according to typical practice at individual UK centres but encouraged to be within 72 hours of admission. Percutaneous coronary intervention (PCI) and coronary artery bypass grafting (CABG) were performed according to current international guidelines.Randomisation was performed via either a secure centralised web-based or telephone assisted system provided by Patients were invited to provide verbal consent to participate in the study after being read an abridged consent form prior to randomisation. This was to avoid delay in those participants that were subsequently allocated to a very early invasive strategy. In addition to the baseline hs-Tn required for study inclusion, samples were obtained post angiography in both trial arms and prior to angiography in the standard of care arm. EQ-5D-5LPrevious high-risk subgroup analyses used the original GRACE score, with a score of >140 stratifying those patients at highest baseline risk. In the TIMACS and VERDICT trials, it was these groups that benefited from an early invasive strategy.Patients at intermediate risk (GRACE 2.0 score ≥90 to<118) were included to attenuate the perceived age bias of the GRACE score, thereby allowing enrolment of younger patients recognised to be at higher risk of future MACE. The following features: anterior ECG changes, ST segment depression, diabetes mellitus on medication and hs-Tn elevation three times the upper limit of normal, have been demonstrated as determinants of poorer prognosis in N-STEMI.24–26Imaging studies confined to N-STEMI are few and primarily descriptive, documenting smaller infarct size than in STEMI.28 29The RAPID N-STEMI CMR substudy applied multiparametric CMR imaging to: (1) assess the impact of the timing of revascularisation on infarct size, volumes and LVEF, myocardial strain, myocardial salvage and extracellular volume and (2) quantify ischaemic burden post-PCI to ascertain whether this can predict risk of future MACE. The RAPID N-STEMI CMR substudy endpoints are listed in Four centres with an established record of high-quality CMR research participated in the substudy. As myocardial injury and infarct size reduces early following MI,33All CMR images will be sent to the core laboratory at the National Institute for Health Research (NIHR) Biomedical Research Centre in Leicester for quality control and central analysis, with the interpreting clinicians blinded to patient information and allocated group.The RAPID-NSTEMI trial will evaluate the effect of a very early invasive strategy on the binary primary composite endpoint of all-cause mortality, non-fatal MI and admission for heart failure (HF) at 12 months following randomisation. The primary and secondary endpoints are listed in RAPID N-STEMI is a superiority trial powered to detect a 25% risk reduction in the primary endpoint. With a two-sided test of α=0.05% and 80% power, 964 patients were required in each arm of the study. Assuming up to 5% withdrawal, 5% crossover and 8% requiring CABG, 1157 patients were planned to be recruited to each group, resulting in a recruitment target of 2314.et al show that at 12 months following N-STEMI the incidence of admission with HF was 14.8%.Sample size calculations were based primarily on the subgroup analysis of GRACE>140 high-risk patients in the TIMACS study. The composite endpoint of death, non-fatal MI and stroke at 6 months occurred in 21.0% of patients in the standard care arm.The CMR substudy is an exploratory mechanistic substudy that had a recruitment target of 200 patients. No formal power calculations were undertaken.A full statistical analysis plan will be completed prior to any analyses being undertaken. Primary analysis will be carried out by intention to treat with imputation for individuals with missing data due to lost to follow-up assessment as having no event. The primary outcome is binary for each individual (yes or no), and therefore, participants experiencing more than one of the composite events will only be counted once. The treatment arms will be compared using mixed effects logistic regression, which will be adjusted for randomisation stratification factors such as hospital site (as a random effect) and GRACE score (as a fixed effect). Treatment comparison estimates will be presented as adjusted ORs and 95% CIs.The analysis of binary secondary outcomes will be carried out in the intention to treat population as per the primary outcome analysis. All other secondary continuous outcomes will be analysed on a complete case approach, where participants will only be included if relevant data are available.Quality of life data (EQ-5D-5L) will be analysed using a mixed effects model with patient as a random effect to account for repeated measures over time. Each patient will contribute up to four postoperative repeated measures to the model. The model will be adjusted for the stratification variables as above. It is expected that some values at later time points will be missing. The mixed effects model specified here will include these patients with partially observed data.An exploratory analysis of the primary outcome in line with the primary analysis plan will be repeated to investigate randomised treatment arm interaction with the following subgroups:Gender: female and male.Age at randomisation: <75 years and ≥75 years.GRACE 2.0 score at admission: >140; >118 and <140; and 90–118.ECG normal versus ECG abnormalities at admission.The primary endpoint will also be analysed as a time-to-first-event outcome. The time will be measured from randomisation and differences between treatment arms compared using Cox’s proportional hazards models, with treatment comparisons presented as hazard ratios and 95% CI. All time to event outcomes will be intention to treat with lost to follow-up censored at date last seen.An exploratory analysis will be conducted repeating the analysis methods of the primary outcome in the efficacy population. The efficacy population excludes individuals that were randomised to early intervention not receiving angiography within 12 hours of randomisation OR were randomised to standard care receiving angiography within 12 hours .The association between CMR outcomes and the primary outcome will be assessed by logistic regression with each CMR variable being included in a separate model. Models will have the clinical outcome as their dependant variable and include the CMR variable as explanatory variable as well as adjusting for treatment arm, site, GRACE score, age and sex.The study is conducted in accordance with the principles of the 1996 Helsinki Declarations, International Conference on Harmonisation-Good Clinical Practice (ICH-GCP) guidelines. The trial has been reviewed and approved by the East of England Cambridge East Research Ethics Committee (18/EE/0222). It is anticipated that data completion will be completed by the end of December 2021, and the study results will be submitted for publication within 6 months of completion.The study was presented to the NIHR University of Leicester Biomedical Research Centre (BRC) patient and public involvement group. Development of the protocol, outcome measures, recruitment to the trial and conduct of the study were discussed. There was a favourable response to the proposed study from the group. Study progress has been fed back to the patient and public involvement group during the course of the trial. Participants are given a study newsletter when they attend their 12-month clinic visit providing information about the study timelines and when the study results will be known. Access to the findings of the study will be made available in a contemporary and user-friendly way and full details of the results provided if the patient requests them.Trial coordination is provided by the Leicester Clinical Trials Unit (LCTU) in collaboration with the chief investigator (CI) and the trial management group. LCTU is responsible for overall trial conduct including data management, quality assurance and statistical reporting. LCTU undertook site initiation visits, database training and ensures all aspects of the trial are performed to the highest ethical and research standards. The study is overseen by a Trial Steering Committee consisting of three experienced clinicians and the CI. An independent data and safety monitoring board convened to provide independent advice on study conduct and safety issues. Clinical events will be adjudicated by an independent Clinical Events Committee.In March 2020, non-COVID-19 clinical research in the UK was suspended as NHS staff and resources were repurposed to frontline services in preparation for the volume of COVID-19 patients expected to place severe pressure on the NHS.The optimal timing of revascularisation in higher-risk N-STEMI is a controversial topic, not least because international guidelines that mandate an early (<24 hours) invasive strategy are not supported by prospective randomised controlled clinical trial data.The major challenges faced due to COVID-19 were threefold. First, significant reductions in admissions with acute coronary syndrome and HF occurred during the pandemic in the UK, with decreases of over to 40% in both disease entities, presumably due to fear of contagion in healthcare settings.Second, the NHS underwent the largest workforce redeployment since its inception to support severely pressurised frontline services treat the vast numbers of COVID-19 patients attending UK hospitals. Research staff were moved to such clinical areas, resulting in all non-COVID-19 related research being left severely disrupted and placed on hiatus until further notice,41Third, and perhaps most fundamentally for RAPID N-STEMI, elective cardiology activity across the UK was effectively cancelled from the beginning of the first wave in March 2020.RAPID N-STEMI is the fifth randomised trial to investigate the timing of an invasive strategy in GRACE score defined higher-risk N-STEMI patients, although the first to specifically investigate this higher-risk population. Prior studies report prespecified GRACE >140 subgroup analyses . FavouraThe choice of trial endpoints is also of note. The recent Early or Delayed Revascularization for Intermediate and High-Risk Non ST-Elevation Acute Coronary Syndromes (EARLY) trial indicated benefit from a very early invasive approach in European Society of Cardiology defined high-risk patients (median GRACE score 122), but such benefit was driven by the softer endpoint of recurrent ischaemic events in a cohort that did not receive P2Y12 inhibitor pretreatment.


However, we here demonstrate that TAD reagents are actually not selective for tyrosine and that tryptophan residues are in fact also labelled with these reagents. This off-target labelling remained under the radar as it is challenging to detect these physiologically stable but thermally labile modifications with the commonly used HCD and CID MS/MS techniques. We show that selectivity of tryptophan over tyrosine can be achieved by lowering the pH of the aqueous buffer to effect selective Trp-labelling. Given the low relative abundance of tryptophan compared to tyrosine in natural proteins, this results in a new site-selective bioconjugation method that does not rely on enzymes nor unnatural amino acids and is demonstrated for peptides and recombinant proteins.Labelling of tyrosine residues in peptides and proteins has been reported to selectively occur  A new strategy for selective tryptophan modification using triazolinedione (TAD) chemistry at pH 4 is shown on peptides and proteins. Additionally, off-target modification of tryptophan residues during the classical TAD-Y click reaction is uncovered. In this regard, tryptophan (Trp) is an interesting target for native conjugation strategies, with an abundance of only just over 1% in proteins.7 Despite the indole side chain not being the most chemically tractable target, several groups have reported methodologies for selective modification of tryptophan in peptides and proteins.8–11 Many of these strategies employ transition metal catalysed reactions and/or conditions limiting downstream biochemical applications. These reactions are typically alkynylations and C–H arylations of the indole.12–16 Also, Trp sulfenylation was demonstrated for peptide ligation.17 While Francis and co-workers showed rhodium carbenoid-based Trp labelling at mild pH,18 this method is dependent on transition metal catalysis and requires long reaction times. An organoradical Trp conjugation was demonstrated on peptides and proteins19 and even if the method is devoid of transition metals, it requires acidic conditions and is not compatible with aqueous buffers. Recently, a novel biomimetic approach for the selective conjugation of tryptophan was developed, the original method however employs UV irradiation and needs to be performed in absence of oxygen.20 This approach was further refined and now allows for the use of visible light in presence of ambient air.21 In 2010, Barbas and co-workers reported a click like reaction for the more abundant tyrosine  using triazolinedione chemistry,22 after which many applications and refinements for protein conjugation followed.23–29 Interestingly, when exploring this powerful Tyr click reaction on Trp containing peptides, we observed a high degree of off-target labelling on Trp residues, even in aqueous buffers. In the initial paper reporting the development of the TAD tyrosine click like reaction, Barbas and co-workers reported that the TAD reaction is amino acid selective for tyrosine in aqueous buffers. In this work we prove that this is in fact not the case and we demonstrate how competitive Trp-labelling remained under the radar for over a decade  to therapeutic development (antibody–drug conjugates).a decade . We show30 Lowering the pH will effectively decrease the amount of tyrosine-phenolate form and thus decrease the extent of reaction of Tyr with TAD. This was further confirmed using additional peptides  was selected for electron transfer dissociation (ETD) MS/MS analysis. In 29 The authors report a very elegant approach to interrogate the amino acid selectivity of a wide range of electrophiles. But as they indicate in their report, potential MS/MS lability of modified amino acids during HCD-based peptide fragmentation was not included in their analysis. This entails that the TAD-tryptophan modification can be missed in such studies. We performed a proteome-wide selectivity study with PTAD alkyne 2c reacting with a tryptic digest of a HeLa cellular proteome in PBS buffer at pH 4 or pH 7.2 in triplicate  was profiled.32 Two recombinant alphabodies were used; the valentine alphabody containing no tryptophans and three tyrosines, and alphabody 586D containing one tryptophan residue next to three tyrosines. A competition experiment between the valentine alphabody and tryptophan containing peptide 1c , the most prevalent risk factor of sporadic Alzheimer's disease,fication . After tet al.24) was performed. Indeed, when taking the Trp-TAD modification into account the data shows that besides the reported Tyr-TAD modifications, also a Trp-TAD modification was abundantly present in the sample (ESI Section S3.1). Furthermore, in the initial study of Ban et al. TAD-modification of a peptide, crucially containing tryptophans and no tyrosine residues, was observed upon myoglobin labelling. In a study by Hu et al. A small amount of modification on tryptophan was observed even in the MS/MS analysis of a TAD modified CRM195 protein.26 Since the TAD modification on tryptophan is largely lost during most MS/MS analysis methods, this was likely originating from a richly tryptophan-modified peptide causing enough traces of the TAD-tryptophan modification to survive MS/MS fragmentation to allow detection. These findings demonstrate that researchers may miss TAD-tryptophan modifications in the analysis of TAD protein modifications.These findings prompted us to look in more detail to earlier reports on the tyrosine click protein modification, and especially how an important off-target effect was able to remain unnoticed for over a decade. Careful reinterpretation of the MALDI-TOF MS spectra obtained from a BSA sample labelled with butyl-TAD followed by a protein digest  chemistry for putative tyrosine-selective protein conjugation, a technique which is growing in popularity. The reversibility of the TAD-tryptophan adducts under MS/MS analysis conditions, in combination with the low abundance and low accessibility of tryptophan side chains likely caused this off-target effect to have remained under the radar. We have found that an exposed tryptophan is in fact kinetically favoured over tyrosine in certain conditions. Lowering the buffer pH further enhanced the selectivity resulting in a transition metal free, buffer-compatible selective labelling method for tryptophan. Thus, in addition to a better understanding of the factors that govern the click-like TAD-based protein conjugation, its scope has been expanded for peptide and protein substrates. The implementation of Trp-substitutions at protein surfaces or loops can thus be an interesting rational design strategy for fully site-selective labelling of native proteins, given they can withstand treatment at pH 4.via ProteomeXchange with identifier PXD031607.Proteomic data are available The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.There are no conflicts to declare.SC-013-D1SC06942J-s001


The findings indicate that as categorized social ability decreases, neural responses to real eye-contact in the right dorsal parietal region also decrease consistent with a neural correlate for social characteristics in ASD.Reluctance to make eye contact during natural interactions is a central diagnostic criterion for autism spectrum disorder (ASD). However, the underlying neural correlates for eye contacts in ASD are unknown, and diagnostic biomarkers are active areas of investigation. Here, neuroimaging, eye-tracking, and pupillometry data were acquired simultaneously using two-person functional near-infrared spectroscopy (fNIRS) during live “in-person” eye-to-eye contact and eye-gaze at a video face for typically-developed (TD) and participants with ASD to identify the neural correlates of live eye-to-eye contact in both groups. Comparisons between ASD and TD showed decreased right dorsal-parietal activity and increased right ventral temporal-parietal activity for ASD during live eye-to-eye contact  and reduced cross-brain coherence consistent with atypical neural systems for live eye contact. Hypoactivity of right dorsal-parietal regions during eye contact in ASD was further associated with gold standard measures of social performance by the correlation of neural responses and individual measures of: ADOS-2, Autism Diagnostic Observation Schedule, 2 Autism spectrum disorder (ASD) is a complex neurodevelopmental condition including distinct behavioral, communicative, and social responses such as reluctance to make eye contact during natural social interactions . The undFor example, in the case of visual sensing and low-level visual processes, variations in patterns of eye-gaze within the population with ASD are well documented –10. ReduTemporal cortex is activated specifically during viewing of eye and mouth movements in the TD population , 22–24. Dynamic and reciprocal face interactions are primary sources of social information. In natural real-life situations, humans typically perceive complex facial information conveyed by facial expressions within very brief exposures including subliminal presentations of fearful faces . HoweverIn response to this knowledge gap, recent developments in functional near-infrared spectroscopy (fNIRS) have applied hyperscanning  to pave the way for much-needed studies of live interactions between individuals and investigations of neural substrates associated with atypical social interactions. The dual brain paradigm provides the infrastructure to quantifiably investigate dyadic behaviors. One potential advantage of two-person paradigms is the application of neural coupling as a measure of dyadic interaction hypothesized to represent sensory and motor processes during cross-brain information exchanges . Howeverin vivo eye contact in both ASD and TD, and to examine their relationship to social performance. The overarching hypothesis is that interactive face processing, with eye contact being a central component, engages high-level demands for rapid interpretation of subtle eye movements and facial cues that convey social meaning. These cues are not included in non-interactive and conventional simulated face stimuli. Thus, it is expected that neural processing of interactive faces in ASD and TD groups will include social systems [The current investigation aims to identify neural systems responsive to  systems ; interac systems –45; and  systems  hypothesnd Edition, ADOS-2) [TD and adults with ASD were compared during real gaze at the eyes of a same-sex lab partner to gaze at a comparable dynamic face video (instructions were to gaze at the eyes under both conditions). In the real-person interaction condition , partners viewed each other’s faces directly while sitting across a table from one other. Findings were compared with a condition in which participants and their lab partners both viewed size-matched faces displayed on a video monitor (Video Eye). The contrast between these two conditions and direct comparisons between ASD and TD groups tested the hypothesis that neural processes responsive to real eye-to-eye contact are altered in ASD relative to TD. Eye tracking and pupillometry during these conditions were used to test the related hypothesis that gaze characteristics  and an autonomic indicator of arousal (pupil size variation) also varied during face processing between TD and ASD. The social performance for all participants with ASD was assessed by clinical interview, including administration of the Autism Diagnostic Observation Schedule (2 ADOS-2) . Self-re ADOS-2) . A goal nd Edition   whose d (ADOS-2 )  and were paid for their participation. Assessment of the participants with ASD capacity to give informed consent was provided by a consensus of trained professional staff who monitored the process and confirmed verbal and non-verbal responses. In order to assure that participants were comfortable during the experimental procedure, participants with ASD were accompanied at all times by a member of the clinical team, who continuously evaluated their sustained consent to participate.nd Edition , and seWASI-II; ); Broad WASI-II; ); SocialWASI-II; ); Beck AWASI-II; ); State-WASI-II; ); and thWASI-II; ). See S3Participants were escorted from the clinical environment to the research environment for fNIRS / eye tracking experiments. An investigator was present during the data acquisition and monitored signs of discomfort during the experiment. All participants were paired with a same-gender TD lab partner. One male (27–28 years old throughout data collection) and one female (22–23 years old throughout data collection) served as lab partners throughout the entire study. Lab partners were not informed of the participant’s group membership before the experiment.Dyads (participant and a gender-matched lab partner) were seated ~140 cm across a table from each other and were fitted with an extended head-coverage fNIRS cap. Each participant was instructed to look straight ahead either at their partner or at a monitor with a video face adjusted in size to subtend the same visual angles as the real face . In the The order of runs was randomly sequenced between viewing a real partner directly or viewing the visual-angle corrected video partner on a 24-inch 16x9 computer monitor placed back-to-back between participants, including a partition to assure that dyads could not see their real partner during video conditions. The face and distance of the video stimuli were calibrated to subtend identical degrees of visual angle in the field of view of the participants and the timing and range of motion of eye movements between partners were the same in both tasks. The time-series and experimental details were similar to prior studies , 45 and At the start of each task, an auditory cue prompted participants to gaze at the eyes of their real or recorded partner. Subsequent auditory tones alternatingly cued eye gaze between eyes or LED according to the protocol time series. The 15-second active task period alternated with a 15 s rest/baseline period. The task period consisted of three 6-second cycles in which gaze alternated on the partner for 3 s and then on a lighted LED to either the right or left  of the participant for 3 s for each of three events. The time series was performed in the same way for all runs. The order of runs was counterbalanced across pairs of participants. During the 15 s rest/baseline period, participants focused on the lighted LED, as in the case of the 3 s periods that separated the eye contact and gaze events. The 15 s activity epoch with alternating eye contact events was processed as a single block.The experimental paradigm  employedTwo Tobii Pro x3-120 eye trackers , one per participant, were used to acquire simultaneous eye tracking data at a sampling rate of 120 Hz. Eye trackers were mounted on the table facing each participant. Prior to the start of the experiment, a three-point calibration method was used to calibrate the eye tracker on each participant. The partner was instructed to stay still and look straight ahead while the participant was told to look first at the partner’s right eye, then left eye, then the tip of the chin. The same calibration procedure for video interactions was performed before recording on a still image presented on the monitor 70 cm in front of the participants. Similar live calibration procedures have been used successfully in prior investigations of in-person social attention , 58. As Eye tracking provided a measure of compliance with task instructions to fixate on the eye as illustrated in Functional NIRS signal acquisition, optode localization, and signal processing, including global mean removal, were similar to methods described previously , 60–65 aOptodes consisting of 40 emitters and 40 detectors were arranged in a custom matrix, providing a total of 54 acquisition channels per participant. The specific layout with the coverage of the optode channels is shown in Eye tracking information including pupil size was exported from the Tobii system to a data processing pipeline and custom MATLAB scripts in where acquired data were used to calculate the mutual eye contact events, accuracy, latency to targets, and pupil diameters. Eye-tracking data were not usable on 5 out of 17 participants with ASD and 4 out of 19 TD participants due to either calibration or equipment problems  intervals to characterize participant eye contact behavior. The eye tracking source was the horizontal component of post-processed trajectories converted to units of arc length (tenths of a degree). There were 1350 observations of 27 participants . To avoid possible inclusion of the large movements into and out of the valid range, the first and last 200 ms of each 3 s eye contact interval were excluded. Three measures were obtained from each interval: Dwell Time, the number of valid retained samples per interval normalized by sampling rate (seconds); Gaze Variability, the standard deviation of the samples centered over each interval, normalized by the number of retained samples ; and pupRaw optical density variations were acquired at three wavelengths of light , which were translated into relative chromophore concentrations using a Beer-Lambert equation –79. SignMethods for calculating neural coherence (synchrony) are the same as those used in previous experiments , 64, 85.fmri.org.Custom code will be provided upon request at <0.05). No evidence for differences was observed between groups during gaze at the eyes in a video face suggesting increased autonomic responses to real faces, but not video faces, in ASD [Even though eye-to-eye contact is often reduced in individuals with ASD, in this investigation, we asked our participants to look directly at the face of the lab partner and to make eye-to-eye contacts during the cued 3 second periods. The recorded measures of gaze time in the “eye box” did not differ systematically between TD and participants with ASD for either the Real Eye or Video Eye conditions see  Figs, co, in ASD .A test of the alternative neural pathways hypothesis for live and interactive face processing in ASD is shown in A further test of the ventral vs dorsal alternative pathways hypothesis is shown in Neural responses  acquired during eye-gaze were regressed by the individual ADOS-2 scores using the general linear model, GLM. The whole-brain main effect of the eye contact activity modulated by ADOS-2 scores is shown in The individual ADOS-2 scores for each participant  Neural systems within ventral occipital-temporal regions were engaged in ASD, whereas in TD live interactive face gaze was associated with dorsal parietal and lateral prefrontal regions; 2) Variations in oculomotor and visual sensing were observed in ASD, including increased positional variation in eye fixations and increased pupillary reactions to live faces, suggesting that visual acquisition factors may also contribute to live face processing difficulties in social signaling; 3) A biological basis for social performance associated with ASD is suggested by the association between ADOS-2 and SRS-2 scores and the counter-correlation of neural responses in the right dorsal parietal regions during real eye-to-eye contact; and 4) Cross-brain synchrony observed for TD real eye-to-eye contact was not observed for ASD, suggesting a reduction in the cross-brain transmission of shared face information. This finding is consistent with the prediction of the dialectical misattunement hypothesis of an interactive mismatch between persons .nd Edition) in ASD and self-report  in both ASD and TD to test the hypothesis that individual differences in social function are predicted by neural responses associated with live eye-to-eye contact.Differences in social performance, including reduced eye contact, are common characteristics of ASD. Although disparities in face processing and oculomotor variations in ASD are well documented, it is not known how these behaviors are linked to the underlying neurophysiology associated with live and natural interactions. In this investigation, we employed a two-person paradigm using functional near-infrared spectroscopy (fNIRS) to acquire neuroimaging responses during live dynamic eye-to-eye contacts with a lab partner that are a close proxy to the behaviors under investigation. Simultaneous eye-tracking and oculomotor responses were also acquired in 17 adult ASD dyads and 19 closely matched TD adult dyads. Social performance was quantified by clinical interview  and right ventral occipital-temporal activity in the case of ASD (ASD > TD). Neural findings modulated by real eye-to-eye contact behavior revealed similar findings of increased right dorsal parietal activity for TD and alternatively increased right ventral parietal activity for ASD consistent with the hypothesis of dorsal eye processing systems for TD and ventral eye processing streams for ASD groups. Individual ADOS-2 scores were negatively correlated with individual fNIRS beta values  within clusters in the right dorsal parietal stream including somatosensory cortices, angular gyrus, and supramarginal gyrus. Similarly, SRS-2 scores for the combined ASD and TD groups were negatively correlated with somatosensory cortices and the supramarginal gyrus also located in the right dorsal parietal stream. Since these two clinical measures provide similar information, it is expected that their relationship to underlying neural correlates would be similar. Neural responses in the right dorsolateral prefrontal cortex (DLPFC) during live eye-to-eye contact were also negatively correlated with ADOS-2 scores. These correlations between social function and the neural responses during live eye-to-eye contact are consistent with a model of hypoactive dorsal parietal and dorsal lateral prefrontal cortex in ASD.Pupillometry, a presumed measure of activity associated with the locus coeruleus-norepinephrine system  as well A natural in-person encounter typically involves active visual sensing of dynamic face landmarks . GuidancThe observed increased positional variation in ASD eye movements leads to the speculation that information characterizing an interactive face may not have been sufficiently acquired for participants with ASD. The finding that live face processing in the ASD group increased activity in ventral and lateral occipital and temporal regions, rather than dorsal parietal regions, could be due, in part, to differences in visual sensing. The observed ventral processes are more consistent with non-interactive face functions than with interactive functions. For example, topographical maps associated with regional specializations for coding simulated faces are well-established. The ventral-occipital cortex is highly selective for and sensitive to pictures of faces , 96. RegNeuroimaging based on functional near-infrared spectroscopy (fNIRS) enables simultaneous acquisition of hemodynamic brain signals from two individuals (hyperscanning) dynamically engaged in natural interactions. Eye tracking acquired simultaneously on both participants during face-to-face engagement enables identification of eye contact events that occur between the partners. The aim to understand the neural mechanisms that underlie social function in ASD has motivated this multi-modal application of fNIRS and eye tracking. In spite of the biological significance of live interpersonal interactions for survival and social well-being, the underlying neural processes of interactive behaviors are relatively novel targets of investigation for natural settings as well as clinical, developmental, and psychiatric applications –34, 98. Current models of face and eye processing in TD and ASD are based primarily on non-interactive paradigms where data are acquired in single-subject situations using conventional stimulus and response models rather than dyadic paradigms that include live social interactions. Understanding neural activity during natural interactions is especially critical in ASD, as the defining social and communicative characteristics of the condition are often attenuated or absent during explicit laboratory tasks . The impThis long-standing experimental paucity of two-person interactive experimental paradigms in ASD, in part, reflects the historical limitations of conventional neuroimaging methods. For example, in functional magnetic resonance imaging (fMRI) solitary confinement in the bore of a scanner with minimal tolerance of head movements constrains/contraindicates investigations of natural, two-person interactions. Fortunately, however, these particular limitations are substantially resolved by recent developments of optical neuroimaging, functional near-infrared spectroscopy (fNIRS), a non-invasive spectral absorbance technique that detects changes in blood oxygen levels in both oxyhemoglobin and deoxyhemoglobin using surface-mounted optical sensors –111. FunThis application of two-person neuroimaging technology to investigate the relationship between the neural underpinnings of interactive face and eye contact and social performance in ASD addresses a prominent and understudied question. Individual clinical evaluations of social performance applied as a second level covariate on whole-brain neuroimaging findings acquired during live real person eye-to-eye contacts confirm a negative relationship between test scores and neural signals in brain regions responsive to real eye-to-eye contacts. Participants with higher ADOS-2 scores, reflecting reduced social performance, showed lower neural signals  in brain regions previously associated with social activity, interactive face processing, and motion sensitivity. Findings also included the right dorsolateral prefrontal cortex, a region implicated in both ASD and commonly co-occurring conditions, such as major depressive disorder. Further, a similar finding was observed for the SRS-2 when the scores of both TD and participants with ASD were combined for regions within the dorsal stream but not the DLPFC. That is, as individual social ability decreased as indicated by the elevated SRS-2, the neural signal decreased in the right dorsal-stream regions. A similar finding was observed for SRS-2 scores and the relationship to neural signals acquired by fMRI in the fusiform gyrus and the amygdala during static face processing . InteresUnique features of this study include the live interactive eye-to-eye task as well as the eye-tracking documentation of compliance combined with the individual difference approach to characterize single participants by both measures. All data were acquired during the epochs when participants directed their eye gaze to within the eye-box of the lab partner. Continuous monitoring confirmed high levels of compliance in all cases. That is, when asked to perform the task, participants were able to do it although eye-to-eye contact was not necessarily a comfortable task for them.These findings of a negative association between right dorsal regions and social performance do not imply a causal role between neural substrates and reduced social function. However, it can be concluded that the dorsal regions found to be related to symptomatology , are involved in the underlying neural conditions relevant to ASD. Given a well-recognized need for biomarkers for ASD that associate with the clinical phenotype at the individual level, the strong relationships observed between neural activity and both clinician and self-reported social function suggest potential utility in key contexts of use, such as stratification for enrichment of clinical trials .In summary, these findings highlight the right dorsal stream system and interactive face processing as regions and tasks of interest for understanding the underlying neural mechanisms that distinguish participants with ASD and TD participants. The specificity of these findings opens new directions for investigating these brain-to-behavior linkages. For example, these regions have previously been implicated in motion sensitivity  and raisWithin this framework, our findings are consistent with the hypothesis that eye movements  during live eye contact and social processing are altered in ASD and suggest that “bottom-up” factors may impact live eye-to-eye interactions. Altered incoming information due to visual sensing factors such as increased fixation variability could fail to capture higher-order motion cues associated with face and social interactions. Individuals with ASD show raised thresholds for the perception of coherent motion , 10, 115The advantages of fNIRS are balanced by technical limitations relative to fMRI. The spatial resolution of fNIRS (approximately 3 cm) does not allow for discrimination of small anatomical differences in functional activity between gyri, and the origin of acquired signals does not extend below the superficial grey matter of the cortex of about 1.5–2.0 cm. Thus, findings of this, and other investigations based on fNIRS technology, are restricted to superficial cortical networks. Although the eye-to-eye contact occurs within a live context which is a novel advance, the gaze context in the study is relatively constrained which is necessary for experimental control and thus also sets constraints on the investigation of naturally occurring behaviors.Despite recent advances in second-person neuroscience, the neurosocial mechanisms that underlie live face processing, a primary social communication system in humans, remains poorly understood. This knowledge gap is particularly profound in autism spectrum disorder where social difficulties are often disabling. Here we apply a two-person dynamic paradigm that incorporates oculomotor, neural, and social metrics as well as cross-brain coherence and individual differences to isolate neural biomarkers associated with reduced sensitivity to real-eye contact. Findings highlight the role of hypoactivity of right dorsal parietal and frontal systems in reduced sensitivity to live faces in ASD.S1 FigColors indicate the percentage of time eye gaze is within the eye region of the partner (dark blue = 0% and bright yellow = 100%) during each epoch of the time series (x-axis). The vertical axis includes all participants with ASD for whom eye tracking data were acquired.(TIF)Click here for additional data file.S2 FigColors indicate the percentage of time eye gaze is within the eye region of the partner (dark blue = 0% and bright yellow = 100%) during each epoch of the time series (x-axis). The vertical axis includes all TD participants for whom eye tracking data were acquired.(TIF)Click here for additional data file.S1 Tablend Edition (WASI-II) was administered to estimate full-scale intelligence quotient scores based on four subtests (FSIQ-4). *Indicates data were not acquired.Assessment measures include the Autism-Spectrum Quotient test ; Broad Autism Phenotype Questionnaire ; Social Responsiveness Scale, Second Edition ; Beck Anxiety Inventory ; State-Trait Anxiety Inventory ; Liebowitz Social Anxiety Scale ; and the Autism Diagnostic Observation Schedule . The Wechsler Abbreviated Scale of Intelligence, 2(TIF)Click here for additional data file.S2 Tablend Edition (WASI-II) was administered to estimate full-scale intelligence quotient scores based on four subtests (FSIQ-4). *Indicates data were not acquired.Assessment measures include the Autism-Spectrum Quotient test ; Broad Autism Phenotype Questionnaire ; Social Responsiveness Scale, Second Edition ; Beck Anxiety Inventory ; State-Trait Anxiety Inventory ; and the Liebowitz Social Anxiety Scale . The Wechsler Abbreviated Scale of Intelligence, 2(TIF)Click here for additional data file.S3 TableJournal of Autism and Developmental Disorders, 43(11), 2584–2603).Groups were similar in terms of age and handedness; however, the ratio of male to female participants was higher in the ASD group than in the TD group. The gender composition of the ASD group is consistent with the estimated 4:1 male:female ratio of ASD diagnosis. This ratio increases to 6 males diagnosed with ASD for every 1 female in people whose cognitive functioning is within or above normal limits, such as those in our sample . A review of the role of female gender in autism spectrum disorders. (TIF)Click here for additional data file.S4 TableNo evidence was found for differences between the groups for FSIQ-4 ; State-Trait Anxiety Inventory (state anxiety items only); or the Liebowitz Social Anxiety Scale, and is taken as evidence in favor of matched groups with respect to these metrics.(TIF)Click here for additional data file.S5 TableGroup averages and individual median percentages of eye-gaze time within the eye box of partners for typically developed (TD) participants (left column) and participants with autism spectrum disorder (ASD) (right column) during the Real Eye Condition. A t-test of these median percentages shows t(25) = 0.28 n.s. See (TIF)Click here for additional data file.


Brassica oleracea or triple-cross hybrids from within or between botanical varieties, the only commercialised latter type being between marrow-stem kale and Brussels sprouts. Eight botanical varieties (15 cultivars) and 13 types of hybrids (50 hybrids) between them were produced and assessed for forage traits in SE Scotland in the early 1980s when there was government funding in Great Britain for such work (terminated in 1990). These previously unpublished results may now be of interest to a new generation of commercial forage brassica breeders. In addition to height and dry matter yield and content, quality traits, such as digestibility and antimetabolites, were assessed. The hybrids with marrow-stem kale as one parent varied in height, but combined a high-dry-matter yield with desirable quality traits for a forage crop. None was ideal and none had a superior combination of traits to the hybrids with Brussels sprouts. The hybrids between marrow-stem kale and January King cabbage were the shortest and a possible alternative to dwarf thousand-head kale. The results can be used to justify new forage brassica breeding programmes.At present, forage kale cultivars for feeding cattle and sheep are either open-pollinated ones from population-improvement schemes within suitable botanical varieties of  Brassica oleracea L.) is grown as a forage crop in Great Britain (GB) and other countries with a similar climate . It combines the high-dry-matter yield of marrow-stem kale with the winter hardiness of Brussels sprouts  and could be used to compare a particular set of four hybrids (2 × 2) with their four parents (2 + 2), with the exception of WC × MS for which there was just one WC parent and hence two hybrids.B. oleracea. The results should prove useful to forage kale breeders in deciding the type of kale to breed, despite being based on just one autumn trial with single-row plots. The growing season was fairly typical for SE Scotland with no abnormal stress conditions and hence good plant growth. However, the winter proved too mild to assess winter hardiness. Nevertheless, the high-dry-matter contents (>17%) of the curled kales, Brussels sprouts and their hybrids can be taken as indicators of winter hardiness.The traditional types of forage kale grown in GB were the tall marrow-stem type for dairy cattle in the autumn and the dwarf thousand-head type for sheep during the winter. Two new types were bred in the 1960s and 1970s at the former Plant Breeding Institute in Cambridge . Both re- contents. Hence, the two January King cultivars included in the current experiment are not typical of cabbages in general, but stand out as having the highest crude-protein, SMCO and SCN- contents of the 21 groups in The two tall marrow-stem kales, the two dwarf thousand-head kales and the two curled kales were included in an experiment conducted by Bradshaw and Borzucki  to asses- contents. This is a good profile for a forage crop, apart from the crude-protein content, which was the lowest (12.6%) of the 13 hybrids. Watts ½, where MS is the mean square for the appropriate ‘error’ variation and N1 and N2 are the numbers of values contributing to each of the means being compared.Analyses of variance were performed and the standard errors of the differences between two means (SED) of interest were estimated as [MS(1/NBrassica oleracea had marrow-stem kale as one parent. These varied in height but combined a high-dry-matter yield with desirable quality traits for a forage crop. None was ideal and none had a superior combination of traits to the already commercially established hybrid with Brussels sprouts. The hybrids between marrow-stem kale and January King cabbage were the shortest and a possible alternative to dwarf thousand-head kale. The results can be used to justify new forage brassica breeding programmes. Suitable starting material could result from pre-breeding in which inbred lines are produced from currently grown open-pollinated cultivars of marrow-stem kale, and inbred lines of vegetable brassicas, currently being used in commercial F1 hybrid breeding, are assessed for forage traits.Seven out the 13 hybrids between botanical varieties of


Facial paralysis is one of the common problems leading to facial deformation. COVID-19 virus rarely has been shown to be associated with facial palsy. Here we present a case of a 60-year-old woman who presented with features of left lower motor facial palsy signs along with common features suggestive of COVID-19 infection. Brain imaging did not reveal any pertinent pathology but her polymerase chain reaction for COVID-19 was positive. This case highlights the fact that acute COVID-19 infection can be considered a cause of motor neuron facial palsy in the ongoing pandemic of COVID-19. Cases with neurological features suggestive of facial palsy therefore should be suspected of acute COVID-19 infection based on other pertinent findings of COVID-19 infection and thus polymerase chain reaction testing should be done. Along with other modalities to investigate facial nerve disorders, testing for COVID-19 infection should also be taken into consideration in selected patients. Peripheral facial palsy should therefore be added to the spectrum of neurological manifestations associated with COVID-19.2 We report a case of left lower motor facial palsy in a woman who tested positive for COVID-19 via Polymerase Chain Reaction (PCR) test.Facial paralysis is a disfiguring disorder that has a considerable impact on the patient because it results in the loss of facial expression which is most commonly caused by a benign self-limiting inflammatory condition known as bell's palsy (BP).A 60-year-old female presented to the Emergency Department with complaints of facial deviation for the last 1 hour .She also had a fever and non-productive cough for the last 4 days for which she was taking over-the-counter antipyretics and cough suppressants. She had no history of any chronic illnesses like hypertension, diabetes and tuberculosis. Examination of the case revealed the weakness of the left side of her face and forehead and thus deviating her face towards the right side. On initial presentation, the forehead was spared but later after about 6 hours of repeat examination, the forehead on the left side was also involved. Motor and sensory examination of upper and lower extremities were normal. Her vital signs were within normal limits.A non-contrast Computed Tomography (CT) scan of the head was ordered and it did not reveal any underlying haemorrhage. Thus she was started on aspirin 75 mg once daily along with supportive care (paracetamol 500 mg thrice daily and antitussives). Repeat examination after 6 hours revealed no additional neurological findings and hence the diagnosis of bell's palsy was made.Along with other routine investigations, her COVID-19 infection status was examined with PCR of her nasopharyngeal swab which came out to be positive. Chest X-ray did not reveal any lesion in her chest. She was then admitted to the isolation ward. Prednisolone 30 milligrams was added to her medication which was to be continued for 14 days and then planned for a tapered dose of prednisolone. Facial physiotherapy was advised which was to be continued for a week. Eye protective measures with artificial tear drop to be used frequently were also added.On the third day of admission upon re-examination, there was an improvement in her weakness although no active facial movements were present. There was no fever for 24 hours, paracetamol was stopped and other supportive measures were continued.She was reassessed on the 7th day of admission. Slight twitching of muscles was visualised while attempting facial movement. There were no signs of active infection. Repeat PCR for COVID-19 was done. It was negative this time. She was then prepared for discharge from the isolation ward. The steroid was continued along with eye protective measures and advised for physiotherapy of facial muscles.3 Bell's palsy is an acute facial nerve palsy involving lower motor neuron (LMN) leading to weakness on one side of the face without any other neurologic abnormalities on examination.4 The aetiology of Bell's palsy is mostly unknown; however, viral infections such as Herpes Simplex Virus (HSV-1) and Herpes Zoster Virus (HZV) are seen to be associated with it.5 Recent studies have shown emerging cases of bell palsy in patients with COVID-19 infection. According to a study done out of 348088 COVID-19 infected patients, 153 patients had new onset of Bell's palsy. The present analysis found a higher incidence of BP in patients with COVID-19 (0.08%).6 This translates to approximately 82 per 100 000 patients with COVID-19. The rate of recurrent BP in patients with previous BP at the time of COVID-19 diagnosis was 8.6%.6 Patients with COVID-19 infection mainly present with fever, dry cough, dyspnea and myalgia but can also progress to respiratory distress requiring hospitalization.7 However, patients can also present with neurologic symptoms such as headache, dizziness, anosmia and dysgeusia.8 A study done among eight patients with COVID-19 developed peripheral facial palsy during infection. In three patients, facial palsy was the first symptom. Nerve damage resulted in mild dysfunction in five patients and moderate in three. SARS-Cov-2 was not detected in CSF by PCR in any of the samples. Seven out of eight patients were treated with steroids and all patients have complete or partial recovery of the symptoms.2Facial paralysis can be central which results from the involvement of upper motor nerves above the pons or peripheral from involving lower motor nerves below the pons. Our case presented paralysis of the upper face suggestive of the peripheral type which is spared in the central type and is the key differentiating sign.10 These mechanisms can impair and damage facial nerves resulting in bell's palsy.The pathogenesis of neurological involvement in COVID-19 patients is still not clear. Some of the proposed mechanisms include the affinity of viruses towards ACE-2 (Angiotensin Converting Enzyme) receptors. One of the structural proteins of Coronavirus is the spike protein (S protein) which is responsible for attachment, fusion and entry into host cells. S protein has a high affinity toward ACE-2 receptors. The expression of these ACE-2 receptors by glial cells and neurons makes the virus an entry site to the brain and its interaction also results in functional changes in ACE-2 receptors causing disruption of the steady state cytokine axis resulting in pro-inflammatory reaction, vascular endothelial injury and neural damage. COVID-19 virus can also directly invade the olfactory bulb and nerve resulting in central nervous system (CNS) entry and neural damage. Immune-mediated damages are also one of the mechanisms for neural injuries. During COVID-19 infection, there is a surge in proinflammatory cytokines resulting in a cytokine storm which damages the blood-brain barrier (BBB) and activates glial cells resulting in neuroinflammation and functional disruption.11 In our case diagnosis was made clinically with detailed clinical history and neurological examination as the patient had a normal CT scan and did not give permission to perform magnetic resonance imaging (MRI), nerve conduction test and lumbar puncture.The patient with bell's palsy presents with paralysis of one side of the face resulting in loss of facial crease and nasolabial fold, drooping of the corner of the mouth, spilling of food and saliva from the side of the mouth and tearing of eyes.5 In our patient prednisolone 30 mg was started and was gradually tapered after which the patient's symptoms started to improve.Bell's palsy is a reversible condition and is managed with corticosteroids with or without antiviral medication. Administration of corticosteroid early within 3 days of the onset of symptoms have a positive outcome and speedy recovery.The above-presented case describes COVID-19 infection as one of the emerging causes of facial palsy. The treatment modality for this case has no significant difference as compared to conventional treatment of idiopathic facial palsy. The concurrent COVID-19 infection must be ruled out in patients presenting with neurological symptoms and other symptoms suggestive of COVID-19. Proper history taking with examination can be used as a guide to suspect COVID-19 in any patient.


This research work extends the fixed interval smoothing based on the joint integrated track splitting (FIsJITS) filter in the multi-maneuvering-targets (MMT) tracking environment. We contribute to tackling unknown dynamics of the multi-maneuvering-targets (MMT) using the standard kinematic model. This work is referred to as smoothing MMT using the JITS (MMT-sJITS). The existing FIsJITS algorithm is computationally more complex to solve for the MMT situation because it enumerates a substantial number of measurement-to-track assignments and calculates their posteriori probabilities globally. The MMT-sJITS updates a current target track by assuming the joint (common) measurements detected by neighbor tracks are modified clutters (or pretended spurious measurements). Thus, target measurement concealed by a joint measurement is optimally estimated based on measurement density of the modified clutter. This reduces computational complexity and provides improved tracking performance. The MMT-sJITS generates forward tracks and backward tracks using the measurements collected by a sensor such as a radar. The forward and backward multi-tracks state predictions are fused to obtain priori smoothing multi-track state prediction, as well as their component existence probabilities. This calculates the smoothing estimate required to compute the forward JITS state estimate, which reinforces the MMT tracking efficiently. Monte Carlo simulation is used to verify best false-track discrimination (FTD) analysis in comparison with existing multi-targets tracking algorithms. We investigate the multi-maneuvering-target (MMT) tracking problem which attained huge awareness in recent studies. Many MMT tracking systems use infinite resolution sensors such as radar and sonar to track targets in a difficult, cluttered environment . HoweverThe multi-target tracking (MTT) algorithms generate the tracks subjected to the available sensor measurements with consequences in the true track  and the false track (clutter). To discriminate the tracks, a widely used false-track discrimination (FTD) technique was developed which mainly identified the detection of targets to confirm the target tracks . The FTDWe apply the smoothing method to improve the target state estimate in the past scan by using the measurements received from upcoming scans compromising a predefined smoothing-time delay ,13. The We have investigated that the optimal smoothing methods in the MTT environment, such as FIsJIPDA and FIsJITS, face a lot of mathematical complexities due to the procedure of joint data association. Therefore, in this paper, we integrate the FIsJITS feasible joint measurement events (FJEs) with the LM method by considering joint events as modified clutter measurements and develop a new fixed measurement interval smoothing algorithm called smoothing multi-maneuvering-targets based on JITS (MMT-sJITS). This new contribution makes the proposed MMT-sJITS simple in a algorithmic structure with less computational complexity. The MMT-sJITS employ JITS in forward-time direction (fJITS) and backward-time direction (bJITS) separately to fuse their predictions. The MMT-sJITS generates tentative forward JITS (fJITS) tracks and develops a validation gate based on the backward measurements without using the sensor measurements, because the backward measurements are predicted subject to the sensor measurements. In contrast with existing algorithms, the MMT-sJITS does not use joint data association for MMT state estimation. The priori smoothing predictions followed by fusion process are obtained to select the smoothing validation measurements from the sensor set of measurements. These smoothing measurements often consist of the joint measurements scenario; therefore, the weighted posteriori smoothing probabilities of these joint smoothing measurements are evaluated to determine modified clutter measurement densities in the coordinates of the current detected measurement. This calculates both the MMT-sJITS and fJITS track estimates in each scan. Ultimately, both the MMT-sJITS and fJITS track are refined using the smoothing validation measurements. The MMT-sJITS track becomes robust due to fJITS track reinforcement in each scan for MMT situation in clutter. For algorithm implementation, we employ the standard nearly constant velocity kinematic model without utilizing any computationally expensive type of maneuvering models (such as discussed by ). Monte k denotes the scan index) is a random variable and the target existence k; for example, the state prediction propagating from k is expressed by:T denotes a scan time, k by:We have assumed that the target state  IPDA in . The MMTThe measurement set collected by a sensor in n method  that conb denotes the backward scan index and N denotes the last scan index of the fixed interval b, but conditioned on the previous backward scan b;k in forward direction but conditioned on the previous scan k;k, conditioned on k conditioning on the measurements set up to k conditioning on the measurements set This section proposes a new integrated smoothing method which integrates the FIsJITS using a modified clutter measurement density to smooth the multi-maneuvering-targets in clutter . An important contribution of the proposed method is to improve the weakness of the FIsJITS algorithm for MMT in clutter. We have assumed the overlapped measurement interval , which ck in the interval k.rval k:N/, a chi-sb indicates a backward scan. An interval consists of N of the corresponding interval. The backward tracks are initialized using each pair of measurements collected from two successive sets of measurements . The trequation ,25:(3)x¯b.The probability of target existence is a track quality measure with respect to a track ne model ,3, that Each bJITS creates a validation gate in the criteria  so that −e)−0.5γ ,13. The Equation  is used G represents the normalized constant factor that must satisfies:The bJITS creates a cluster of tracks where the measurements are mapped to the tracks using Equation  and formendently . TherefoEquation . Here, fEquation  and G reThe modification in the probabilistic equation (obtained in Equation ) avoids essed by:μb,iτ=ρb,Equation . HoweverEach Simultaneously, each validated measurement forms a new track component with corresponding component existence probability expressed by:b. Similarly, the backward multi-tracks state estimations are calculated recursively using Equations (b.The predicted bJITS track components (obtained from Equation ) are estquations –14) in  in (14)xb arrives at the first scan k scan of an interval k using Equation (When backward scan distance  method tEquation .(15)x¯kThe fJITS creates a validation gate by exploiting backward multi-track components state predictions calculated in scan criteria  are usedquations :(17a)P¯In addition, if any of the bJITS track fails to satisfy Equation , then itequation :(18)ζk|NThe MMT-sJITS calculates a priori smoothing Equation  in:(22)PEquation  modifiesEquation .The MMT-sJITS uses Equation (17) in the validation selection criteria  to find The MMT-sJITS maps feasible outcomes quations  and 25)y˜k,iτ,c quations  replacinSimilar to the bJITS iteration, the MMT-sJITS calculates a posteriori smoothing quations  and 26)τth targequations  replacinThe MMT-sJITS uses Equation (17) in Equation  (replaciEquation ) to obtaEquation ; that isk based on the measurements provided up to scan N in an interval k to compute the MMT-sJITS output track state statistics.However, if Equation  is utiliSince the clusters are already formed in forward path track with the MMT-sJITS update, the steps including measurement selection, FJE formation and posteriori probabilities of joint measurement events are not required for the forward track state estimation. The MMT-sJITS calculates the state estimate of the fJITS track components by utilizing the smoothing component measurements (selected from Equation ) and updEquation  with subEquations  and 30)30) are ak. Each updated fJITS track state component calculates its updated component existence probability by utilizing the Equations (The fJITS track components state estimation such as Equation ) in the Equation . ConsequEquation  for recuquations  and 28)x^k|kτ,c Thus, each fJITS track becomes a powerful tool for treating the bJITS multi-tracks as measurements and associating the backward predictions for fusion in the forward validation gate. This not only improves the fusion of forward and backward validated predictions but also enhance the capability of the smoothing algorithm for an efficient multi-maneuvering-target tracking in the subsequent scans.The proposed MMT-sJITS algorithm is implemented using the fixed overlapped measurement interval, which consists of +1 scans ,22 as ilN) are used to initialize backward tracks . Th. ThNth tquations . For exaapproach ) in everfJITS multi-tracks are initialized using two successive scan measurements . He. Hek conEquation  to obtaiLet the current fixed measurement interval starts from scan In each forward-time scan, the newly intialized fJITS are concatenated with existing fJITS tracks so that both the new and estimated fJITS track components are propagated simultaneously for fusion in each scan. The maximum smoothing is obtained when we overlapped the current fixed measurements interval to the next interval. For example, we have overlapped the interval at quations –14) to  to 5th sThe false track discrimination (FTD), root-mean square error (RMSE), and the statistics of the multi-track retention of the proposed MMT-sJITS method are compared with the existing MTT algorithms such as FIsJITS, JITS, FIsJIPDA, and JIPDA.The MMT-sJITS method applies the track component merging and pruning ,26,27,28Confirmed true tracks (CTTs): To achieve a CTT, a confimed track must satisfy Equation  with γ≤2Confirmed false track (CFTs): With Without loss of generality, a tentative track becomes a confirmed track if its updated track existence probability excelled by the confirmation threshold; or else becomes a terminated track if its updated track existence probability sank by a track deletion threshold . The MMTribution ,13. The We assumed a point target tracking; therefore, at most one measurement belongs to a target track. Therefore, we have utilized the auction algorithm  to identWe assume that two maneuvering targets are moving in the two-dimensional cluttered environment, which is corrupted with random number of clutters with the measurement density of x-axis and y-axis, respectively. A sensor produces a measurement noise covariance The surveillance platform depicted in k. Furthermore, assume that a low initial probability of target existence Each algorithm including the MMT-sJITS assumes the Markov Chain One model ,3 of theAn important conclusion from the simulation results presented in this paper and from the one which was illustrated in FIsJITS  is that ussed in . In addiussed in . In thisussed in . This maussed in  and utilThe track retention statistics of all algorithms are determined by storing the identity number of the CTTs corresponding to each target in scan Case: a number of CTTs followed by a Okay: a number of CTTs pursued the same Swapped: a number of CTTs exchanged by some other target CTT Lost: a number of CTTs in scan 30 lost due to either track deletion because of the low target existence or low component existence probabilities, or they became CFTs;Result: a number of CTTs in the final scan Execution time [s]: the average calculation time per each Monte Carlo Simulation run that was analyzed using the MatLab R2020b software on the platform, 11th Intel Core™i7-1165G7 .In the The MMT-sJITS extends the FIsJITS method in a multi-maneuvering-targets scenario. Compared to existing algorithms, the MMT-sJITS does not use a multi-target joint data association process. In the joint measurement situations, the MMT-sJITS considered the joint cluster-measurements being followed by other cluster tracks as modified clutters, thus avoiding the influence of joint measurement events. The proposed method utilized the modified smoothing clutter measurement density to obtain the smoothed components state estimate and forward JITS components state estimate. The impact of backward-running JITS multi-tracks reinforced the forward multi-tracks to obtain the MMT-sJITS state estimation for an effective MMT tracking. This significantly improved FTD performance and accuracy in the estimation compared to other methods.


Ovotesticular disorder of sexual development (DSD) is the rarest of DSDs with an incidence of 1:20000  and magnetic resonance imaging (MRI) were suggestive of a vaginal pouch. The patient underwent endo-evaluation followed by robot-assisted excision of the vaginal pouch. Endo-evaluation showed two orifices in the posterior urethra. The posterior orifice was leading into a blind-ending rudimentary uterus and the true urethra was lying anteriorly. The DaVinci Xi Robotic Surgical System was used and the entire pouch was dissected free of the surrounding tissues using monopolar scissors. The pouch was transected just a few millimetres from its junction with the urethra. The urethra was then closed with V-loc 4-0 suture. The patient was discharged on postoperative day 2 and the catheter was removed on day 21.Follow-up VCUG at 6 weeks did not show any residual pouch. There was no complaint of post-void dribbling or UTI at 30 months of follow-up.Robot-assisted laparoscopy should be considered as an alternative to laparoscopy for the primary treatment of a large symptomatic vaginal pouch.


Cyclic peptides are an attractive option as therapeutics due to their ability to disrupt crucial protein–protein interactions and their flexibility in display type screening strategies, but they come with their own bioanalytical challenges in metabolite identification. Initial amide hydrolysis of a cyclic peptide results in a ring opening event in which the sequence is linearized. Unfortunately, the mass of the singly hydrolyzed sequence is the same (M + 18.0106 Da) irrespective of the initial site of hydrolysis, or soft spot. Soft spot identification at this point typically requires time-consuming manual interpretation of the tandem mass spectra, resulting in a substantial bottleneck in the hit to lead process. To overcome this, derivatization using 2-pyridine carboxaldehyde, which shows high selectivity for the alpha amine on the N-terminus, was employed. This strategy results in moderate- to high-efficiency derivatization with a unique mass tag and diagnostic ions that serve to highlight the first amino acid in the newly linearized peptide. The derivatization method and analytical strategy are demonstrated on a whole cell lysate digest, and the soft spot identification strategy is shown with two commercially available cyclic peptides: JB1 and somatostatin. Effective utilization of the automated sample preparation and interpretation of the resulting spectra shown here will serve to reduce the hit-to-lead time for generating promising proteolytically stable peptide candidates. All data were acquired in centroid format, with full MS in the range of For background subtraction preparation, the acquired LC/MS data were first processed with a Slicer utility in RecalOffline  to extract the AIF data into separate data files. The AIF data files were converted to NetCDF format using the file conversion utilities provided by the instrument vendors. Background subtraction was conducted on the NetCDF format of the AIF datasets. In this proof-of-principle study, where incubation samples were generated with multiple cyclic peptides, the data of samples from other cyclic peptides were used as controls (referred to as “control scans” below) to provide matrix component coverage for the analysis of data of the sample of a given cyclic peptide . The background subtraction algorithm has been described elsewhere ,17,18,19A novel strategy for cyclic peptide soft spot identification that relies on selective derivatization of the newly formed N-terminus after ring opening hydrolysis was presented. The 2PCA method, first proposed by Francis and co-workers for α-amine derivatization under native conditions, was repurposed for in vitro cyclic peptide metabolite identification. By simply adding the 2PCA reagent into our time course quench solution and incubating it overnight, the new strategy fits nearly seamlessly into the automated sample preparation workflow already in place in typical early-stage drug discovery environments. Utilization of the approaches demonstrated herein can potentially decrease the time required for HCOIs to become (in vitro) proteolytically stable candidates from months to weeks. Here, the method was showcased using a tryptic digest of a HeLa whole cell lysate, and then subsequently demonstrated in a real semi-automated time course with two commercially available cyclic peptides, JB1 and somatostatin. Combining the simple N-terminal derivatization with previously optimized background subtraction methods facilitates simple and rapid interpretation for high-confidence soft spot identification. Future work will focus on extending the 2PCA methodology to assay proteolytic stability in vivo.


However, because the difference between number of left-handed and right-handed photons in the free electromagnetic field is less than a percent, there is still a great challenge to measure the difference.Researchers from Jiangnan University, together with their international collaborators present a versatile method to recognize the polarization state of light by using chiral gold nanoparticle film deposited onto anodized aluminum oxide substrates. In integrated photonic devices, the photocurrent is directly related to the polarization state of light with 2.41-fold difference between right-handed circularly polarized and left-handed circularly polarized light, which provides advantages over their inorganic or organic chiral film counterparts by 1–2 orders of magnitude. The concept is clarified referring to the wave-guided chiroplasmonic modes propagating along the film surface and charge entrapment in phenylalanine on gold nanoparticles.


There are specific TTR mutations associated with the development of a polyneuropathy (PNP), while others are predominantly associated with a cardiomyopathy or a mixed phenotype.2Amyloidosis is a heterogenous group of diseases characterized by progressive organ dysfunction caused by deposition of misfolded proteins in various target organs including the heart and the nervous system. The two most common types of amyloidosis that affect the heart are immunoglobulin light chain cardiac amyloidosis (AL-CA) and transthyretin cardiac amyloidosis (ATTR-CA). ATTR-CA is an infiltrative cardiomyopathy that arises secondary to deposition of insoluble transthyretin amyloid fibrils in the extracellular space of the myocardium. The natural history of ATTR-CA can be variable, but if untreated is characterized by progressive heart failure, conduction system disease, arrhythmias, and ultimately death, which typically occurs only several years after diagnosis.4 Now in the era of available therapeutic agents8 that can slow or halt disease progression, the early identification of patients with ATTR-CA has become increasingly more important than ever. Technetium-labeled cardiac scintigraphy  plays a critical role in the non-invasive diagnostic pathway for patients with suspected ATTR-CA.9 Advances in bone scintigraphy coupled with increased awareness have led to an exponential increase in the number of patients diagnosed with ATTR-CA. Furthermore, no longer requiring an endomyocardial biopsy to make the diagnosis and instead in many cases being able to non-invasively diagnosis ATTR-CA in appropriately selected patients  has contributed to increasing diagnoses.11Historically considered a rare disease, cardiac amyloidosis is currently recognized as a significantly more prevalent clinical entity.12 In contrast to patients with AL-CA, patients with ATTR-CA had both cardiac uptake as well as extensive soft tissue uptake in the shoulder, gluteal, and abdominal wall muscles. A later study by the same group reported higher soft tissue to femur ratio in relation to Perugini grade using DPD scintigraphy in patients with ATTR amyloidosis.13In addition to providing information about possible cardiac involvement in patients with suspected ATTR-CA, DPD scintigraphy can also reveal extra cardiac uptake of tracer. Several prior studies have reported characteristic extra cardiac uptake in soft tissues in patients with ATTR amyloidosis. In a study by Hutt et al., a cohort of patients with suspected or proven cardiac amyloidosis underwent DPD scintigraphy.Journal of Nuclear Cardiology, Wollenweber et al. reported the potential utility of increased [99mTc]-3,3-Diphosphono-1,2-Propanodicarboxylic Acid (DPD) soft tissue uptake to serve as an imaging marker that identifies patients with ATTR-CA at a higher risk for having concomitant PNP.14 The authors of this study hypothesized that there would be higher soft tissue DPD uptake in ATTR-CA patients with PNP as compared to ATTR-CA patients without PNP. In this retrospective, single center study, a cohort of 50 patients with ATTR-CA (41 wild-type and 9 hereditary) who had undergone whole body DPD scintigraphy and a nerve conduction study (NCS) were evaluated. The study cohort was predominantly male (80%) with a mean age of 77.4 ± 8.2 years. The diagnosis of ATTR-CA was made on the basis of the absence of a monoclonal protein in the blood or urine10 and a Perugini14 grade 2 or 3 (36 patients), an endomyocardial biopsy (12 patients), or a positive DPD scan and genetic testing (2 patients). NCS results revealed that 50% of patients (19 wtATTR and 6 hATTR) had a sensorimotor PNP, 20% had a sensory PNP (9 wtATTR and 1 hATTR), and the remaining patients (13 wtATTR and 2 hATTR) did not have a PNP. The mean time interval between the bone scan and NCS was 210 ± 93 days. A subgroup of the study cohort (22 patients) also underwent quantitative SPECT/CT of the thorax directly after planar imaging. As SPECT/CT image acquisition was primarily performed over the thorax, the subcutaneous abdominal fat was not available for evaluation in all patients. Therefore, standardized uptake values in the subcutaneous fat tissue of the left axillary region were evaluated instead.In this issue of the The results from the visual DPD bone scintigraphy analysis revealed that the majority of patients with wtATTR (97.6%) and hATTR (100%) had a Perugini grade of 2 or 3. Furthermore, whole body imaging analysis of the entire study cohort demonstrated a significantly higher Perugini score in patients with ATTR-CA and PNP as compared to patients with ATTR-CA and without PNP. Excluding patients with a history of diabetes mellitus from the analysis, further increased the significance of this finding between the group of patients with ATTR-CA with and without PNP. In patients with wtATTR and without a history of diabetes mellitus, there was also a statistically higher Perugini score observed in patients with PNP as compared to those without.The analysis of quantitative DPD bone scintigraphy revealed that even after exclusion of patients with a history of diabetes mellitus, there was not a statistically significant difference between whole body DPD uptake normalized to the applied activity between ATTR-CA patients with and without PNP. Furthermore, region of interest (ROI) analysis revealed that skull uptake was significantly decreased in ATTR-CA patients with as compared to without PNP. This finding was also observed in the subgroup of patients with wtATTR.peak in the left axillary region subcutaneous fat between patients with and without PNP irrespective of history of diabetes mellitus.Regarding the thoracic SPECT/CT group analysis, there was significantly increased DPD uptake in the left axillary region subcutaneous fat in ATTR-CA patients with as compared to without PNP. Soft tissue DPD uptake was still significantly increased in ATTR-CA patients with as compared to without PNP after exclusion of patients with diabetes. In a subgroup analysis of patients with wtATTR, there was not a statistically significant difference in SUV18 The use of quantitative bone-avid cardiac scintigraphy to quantify amyloid burden in ATTR-CA is currently an area of great interest and holds the promise of aiding with early disease diagnosis, risk stratification, prognostication, assessment of treatment response, and potentially improvement in patient outcomes.19 The findings from this current study suggest that a higher Perugini score and increased DPD soft tissue uptake in subcutaneous fat could potentially be a clinically useful diagnostic imaging marker to identify patients with ATTR-CA and PNP. If so, this observation would further expand the diagnostic utility of DPD scintigraphy to identify not only patients with ATTR-CA, but also to potentially identify patients with PNP. Furthermore, this observation underscores a potential added benefit of utilizing DPD over PYP scintigraphy in being able to quantify extracardiac radiotracer uptake in the diagnostic evaluation of patients with suspected ATTR-CA.Although relatively small and retrospective in nature, this study does have merit. Not only does this study contribute to the current ATTR-CA knowledge base, but it also expands the potential diagnostic utility of using DPD scintigraphy in patients with suspected ATTR-CA. The observation that patients with ATTR-CA with PNP have increased soft tissue DPD uptake is in line with prior studies that have utilized quantitative bone-avid radiotracer imaging and have reported a correlation between tracer uptake and cardiac amyloid burden and phenotype.It is however worth mentioning that this study does have several limitations. As previously mentioned, the primary limitation is its small sample size and retrospective nature. Furthermore, the association of a higher Perugini score with PNP observed in this study cannot rule out increased DPD uptake secondary to more advanced disease. Lastly, the extended time interval between the bone scan and NCS, lack of details surrounding the onset of PNP, and not excluding other causes of neuropathy with the exception of diabetes mellitus, represent additional limitations of the study.In summary, ATTR-CA is an increasingly recognized cause of cardiomyopathy and/or PNP. Given the progressive nature of untreated ATTR-CA and the availability of disease modifying therapies, identifying patients early in their disease course remains of critical importance. The use of DPD bone scintigraphy plays an important role in the non-invasive diagnostic evaluation of a patient with suspected ATTR-CA. Furthermore, the findings from this study suggest that increased soft tissue DPD uptake could be used as a potential diagnostic imaging marker to identify patients with ATTR-CA with an increased risk of PNP. Future prospective, randomized control studies are needed to confirm these observations on a larger scale and further clarify the clinical significance of increased soft tissue DPD uptake in patients with ATTR-CA.