Deep learning provides an opportunity to automatically segment and extract cellular features from high-throughput microscopy images. Many labeling strategies have been developed for this purpose, ranging from the use of fluorescent markers to label-free approaches. However, differences in the channels available to each respective training dataset make it difficult to directly compare the effectiveness of these strategies across studies. Here, we explore training models using subimage stacks composed of channels sampled from larger, “hyper-labeled,” image stacks. This allows us to directly compare a variety of labeling strategies and training approaches on identical cells. This approach revealed that fluorescence-based strategies generally provide higher segmentation accuracies but were less accurate than label-free models when labeling was inconsistent. The relative strengths of label and label-free techniques could be combined through the use of merging fluorescence channels and using out-of-focus brightfield images. Beyond comparing labeling strategies, using subimage stacks for training was also found to provide a method of simulating a wide range of labeling conditions, increasing the ability of the final model to accommodate a greater range of candidate cell labeling strategies. I.As high-content, high-spatiotemporal cellular imaging becomes more widespread, the ability to perform cellular segmentation both quickly and accurately becomes increasingly critical for efficient cellular analysis and feature extraction. Advances in deep learning have positioned neural networks as a powerful alternative to traditional approaches such as manual or algorithmic-based segmentation.Adapting deep learning techniques for cellular segmentation, however, presents some unique challenges due to the high levels of incongruity across images of cells. For instance, images of cells may appear significantly different as a result of either the imaging techniquesTraditional approaches to automated cell segmentation from microscope images generally fall into two main categories: fluorescence-based and label-free approaches. Fluorescence-based approaches often boast higher segmentation accuracies but require the addition of fluorescent markers. However, reliance on specific fluorescent markers confers some significant disadvantages as microscopy tends toward multiparametric, high-throughput imaging. Most notably, fluorescence-based segmentation limits multiparametric imaging by dedicating a portion of the fluorescence spectra for segmentation that might, otherwise, be used. Fluorescent markers can also induce stress on the cell, either directly or as a by-product of imaging, and are, therefore, best avoided when possible.supplementary material Fig. S2) to train a robust cell segmentation model. For simplicity, we, henceforth, refer to the source image stack as the hyper-labeled image stack and subsets of images used during training as subimage stacks. Beyond reducing the dataset size, this approach to training confers some additional advantages, including the ability to compare different labeling approaches on an identical dataset. In particular, we use this dataset to explore new approaches to preprocess data entering segmentation models including the use of out-of-focus (OoF) brightfield images and the concept of merging fluorescence channels into a single input channel. To keep the implementation practical, our approach uses fewer than 300 labeled cell examples and can be trained in less than a day on a modern Graphics Processing Unit.As biology moves further into multiplexed imaging, including using localized sensors of the microenvironment, it is often not feasible to sacrifice the bandwidth to an ideal segmentation marker. Thus, we envision using multiple sensors that are biologically relevant but nonideal for segmentation. For instance, genetically encoded sensors expressed in either the cytoplasm or the mitochondria may each be used to help demarcate and mask individual cells. However, the information provided by these signals is very different: cytoplasmic markers can clearly define cell boundaries in isolation but may become indistinct across adjacent cells; mitochondrial markers do not reach the limits of cell boundaries but provide a gap in fluorescence, which can be used to more broadly separate adjacent cells. The primary challenge when using this information is that the combination of auxiliary fluorescence signals available to the segmentation model may vary from experiment to experiment or cell to cell. This requires that a segmentation model is trained to maintain performance across a wide range of potential experimental labeling conditions, including the absence of fluorescence labels of any kind. This would be difficult under the traditional approach to training deep learning models, as accounting for all possible experimental configurations would require collecting and labeling a prohibitively large and expensive dataset. Fortunately, microscope images possess a unique property that may be exploited to substantially reduce this dataset requirement: the channels of microscope images exist as stacks of independent images. This means that a subset of channels from a larger image stack can be assembled to create an entirely new representative microscope image. For example, an image stack of a cell composed of channels capturing a cytoplasmic marker, a membrane marker, a mitochondrial marker, and a brightfield image can be used to simulate a cellular image where only the cytoplasmic marker is present. The ability to create representative image substacks from a larger image stack is in stark contrast to other vision-based image modalities , where removing a specific color channel would produce an image that is no longer representative of the target data. Here, we use source image stacks composed of three fluorescence labels and brightfield images imaged at seven different focal planes to simulate a wide range of expected experimental images , the in-focus brightfield channel (the brightfield model), or a combination of all three fluorescent markers (the fluorescence model), random channels (the random model), or black channels as a negative control . accuracy . Segmentaccuracy . More geaccuracy  was addeaccuracy . As the B.supplementary material Fig. S9). The segmentation of brightfield images can be impacted by focus.μm) and one below  slightly improved performance compared to the in-focus brightfield model . Here, the combination of brightfield and fluorescence resulted in increased segmentation accuracy as compared to either fluorescence or brightfield alone [supplementary material Fig. S8(d)], while maintaining this performance in the face of inconsistent fluorescence labeling [supplementary material Fig. S8(e)]. As with the primary dataset, training the model with a moderate dropout rate produced models that maintained their performance across a wider range of validation dropouts [supplementary material Fig. S8(f)].To determine whether the benefits of combining a fluorescence merge channel with the brightfield were also beneficial to other cell-based datasets, an external dataset was sourced and labeled as described in Sec. III.The unique nature of microscope images provides many exciting opportunities for innovation when adapting deep learning techniques for cell segmentation. Notable among them is the independence of channels within a microscope image stack, such that representative subimage stacks can be generated from a larger source, or hyper-labeled, image stack . TraininFluorescence-based approaches boast strong accuracies for fully labeled cells , but perThe ability to simulate various labeling conditions using subimage stacks was found to be particularly important when training models that are required to segment a more diverse set of experimental setups. For instance, we found that models trained on perfectly labeled cells boasted the highest accuracies but were also the quickest to lose performance as labeling became more inconsistent . In contsupplementary material Fig. S9). Even if the final objective is to create a label-free segmentation model, imaging cells with a cytoplasmic or membrane marker in addition to the relevant brightfield channels allows more precise ground-truth labels to be created than through the brightfield channels alone. The label-free model can then be generated by training the model using subimage stacks containing only the relevant brightfield channels. By extension, combinations of markers may even be used in conjunction with other algorithmic or deep learning segmentation approaches to automate label generation for larger datasets, whether or not those specific markers will be used to train the model.A more subtle advantage of collecting hyper-labeled image stacks for training is that they expedite the creation of ground-truth labels. In certain cases, this may even allow ground-truth labeling to surpass human level performance. For instance, segmenting brightfield images of cells in close proximity is difficult for manual operators, but simple when a cytoplasmic or membrane marker is present (see IV.A.+),μm), with 0 μm representing cells in manual focus. Altogether, these 10-channels were used to form our hyper-labeled image stacks. From these images, 275 fully labeled cells  were manually isolated using a custom Fiji plugin to form our training and validation datasets. Ground-truth segmentation labels were generated in Gimp at the pixel level by two independent operators to minimize inter-operator bias and by alternating between the fluorescence and near-focus (−3 to 3 μm) brightfield channels to minimize biasing performance toward either imaging method. This final dataset represents a combination of 106 AD293 and 169 INS1E cells, with an additional 175 cell-free images added to the dataset as negative control. Of the cellular images, approximately 26% represented isolated cells , while the remaining 74% had at least one neighboring cell in the field of view. Cell images were resized to a final size of 400 × 400 px (9.28 × 9.28 μm2) before entering the model.Cells were labeled with three distinct subcellular markers for the cytoplasm (Turquoise2-tagged Apollo-NADP+), the membrane (YFP-Mem), and the mitochondrial matrix (Mitotracker Deep-Red FM). This dataset was composed of 96 fully labeled cell images and 60 cell-free negative controls.An additional test set was created as described above using cells that were labeled with subcellular markers for the endoplasmic reticulum . To make use of transfer learning, pretrained weights  and S3(b)]. The maximum learning rate was chosen by training the model over 100 iterations while gradually increasing the learning rate from supplementary material Figs. S3(c) and S3(d)]. As training was performed in two parts (5 epochs with the pretrained descending arc frozen and then 100 epochs with the entire network unfrozen), two maximum learning rates were chosen. Based on these results, the maximum learning rate was chosen conservatively to be The majority of hyperparameters used represent best-practice recommendations.supplementary material Fig. S3(e)]. Overall accuracy declined in either direction; however, lower values were associated with fewer background pixels being misclassified as belonging to the cell, while the inverse was true for larger weights . As many cell masking applications would prefer falling slightly short of the cell boundary  over surpassing the cell boundary , a scaling factor of 0.5 was chosen to minimize false positive pixels without significantly impacting overall accuracy.Models were trained using a two-class weighted cross entropy loss,supplementary material Figs. S3(f) and S3(g)]. Significant, normally distributed variability was found in the final training accuracy when the dataset was randomly split. When the dataset was consistently split, the accuracy did not vary. This indicates that it is critical to use identically split training and validation datasets across each model tested during a single experimental replicate. This also suggests that the relative performance of each model is a more useful metric than an absolute segmentation accuracy percentage. Based on these findings, splits were kept consistent across models during a run to reduce the impact of training-validation splits on relative performance.Training and validation sets were divided using a randomized 80:20 split of the cells from the complete dataset. To determine how the split between the training and validation set may impact model accuracy, models were trained on all-black images using either a consistent (100 models) or a re-randomized (100 models) training-validation split [C.supplementary material Fig. S5, examples of brightfield and fluorescence channels were passed through a variety of transforms including brightness and contrast, dihedral transforms, image flipping, image jitter, perspective warping, image rotation, skew, and symmetric warping. Parameter ranges for each transform were chosen that produced realistic cell images for both fluorescence and brightfield images. Despite the unnatural appearance, zero-padding was used when necessary to avoid twin-cell artifacts in the ground-truth labels . Data augmentation beyond a squaring crop and resize was not applied during validation.Significant data augmentation was used to keep the dataset relatively small. As outlined in D.•Simple loading: load a specific channel from the ten-channel source image stack.•Randomization: load a single random channel from a predefined subset of channels.•Channel dropout: perform a randomized test against a dropout percentage. Load the channel normally if passed, load a blank channel otherwise.•Merge: merge the contents from multiple channels into a single channel before loading.•Merge dropout: perform a randomized test against a dropout percentage and only include that particular channel in the merge if passed.Training was performed on three-channel subimage stacks assembled from the ten-channel, hyper-labeled image stacks. Creation of these three-channel subimage stacks was performed during training using model-specific loading codes. These loading codes provided independent, channel-specific instructions to a custom dataloader directing how each of the three channels would be assembled from the source dataset see . The usesupplementary material Fig. S2.A complete representation of the loading combinations used can be found in E.Model performance was determined using their average segmentation accuracy, which was calculated as the percentage of pixels that were accurately classified as compared to the ground-truth label . Unless otherwise noted, line graphs were plotted using a 10-point moving average for clarity (with empty padding for early epochs) and error bars represent the standard error of the mean. Significance was assessed using a paired t-test.F.No ethics approval was required for the work in this paper.supplementary material for additional data on U-Net architecture, segmentation parameters, and test sets referenced in this article.See the


Avr genes from rust pathogens, which has recently begun. Although developed with wheat resistance genes as the primary targets, TEnSeq approaches are also applicable to other classes of genes as well as for other crops with complex genomes.The wheat-rust pathosystem has been well-studied among host–pathogen interactions since last century due to its economic importance. Intensified efforts toward cloning of wheat rust resistance genes commenced in the late 1990s with the first successful isolation published in 2003. Currently, a total of 24 genes have been cloned from wheat that provides resistance to stem rust, leaf rust, and stripe rust. Among them, more than half (15) were cloned over the last 4 years. This rapid cloning of resistance genes from wheat can be largely credited to the development of approaches for reducing the genome complexity as 10 out of the 15 genes cloned recently were achieved by approaches that are summarized as TEnSeq (Target-sequence Enrichment and Sequencing) pipelines in this review. The growing repertoire of cloned rust resistance genes provides new tools to support deployment strategies aimed at achieving durable resistance. This will be supported by the identification of genetic variation in corresponding  Puccinia. Significant yield losses from each of the three diseases have been reported from almost all major wheat-growing regions worldwide. Losses to leaf rust were approximately $350 million between 2000 and 2004 in the United States  identified only in the last 4 years. This recent accelerated progress was made possible by (i) the public availability of the first high quality reference genome for wheat (Chinese Spring RefSeq v1.0) and (ii) the development of various approaches for reducing the genome complexity to allow targeted resequencing analyses. In particular, 10 out of the 15 genes cloned since 2016 were identified through pipelines involving Target-sequence Enrichment and Sequencing (TEnSeq). In this review, we briefly covered some general features of the wheat-rust pathosystem and the most recent progress in the area of cloning wheat rust resistance and rust fungal effector genes.Puccinia graminis Pers.: Pers. f. sp. tritici Erikss. & E. Henn (Pgt), P. triticina (syn. P. recondita Rob. ex Desm. f. sp. tritici) (Pt), and P. striiformis Westend. f. sp. tritici Erikss. & E. Henn. (Pst), respectively , activated by the recognition of microbial or pathogen-associated molecular patterns (MAMPs or PAMPs), and effector-triggered immunity (ETI), which encompasses “gene-for-gene” type of resistance . BacteriHm1 from maize (Zea mays) was the first cloned R gene and encodes an enzyme that detoxifies a toxin from the fungal pathogen Cochliobolus carbonum  is an exception in possessing nearly 1,000 NLRs despite having a relatively small genome (740 Mb). In contrast, the number of NLRs in orchids species (Apostasia shenzhenica) with a genome size of 349 Mb was reported to be normally less than a 100  genome is one of the most challenging plant genomes to study. It is highly repetitive (∼85%) and approximately 15.4–15.8 Gbp in size, which is five times larger than the human genome  and the wild tetraploid progenitor T. diccocoides and cultivated tetraploid wheat T. turgidum cv. durum .The massive and complex genome of wheat has made the isolation of individual genes a challenging task. Among ∼200 rust resistance genes cataloged in wheat, only a small number have been cloned and had their molecular functions studied. A complete list of the rust resistance genes cloned so far from wheat is shown in Various gene cloning strategies have been used to position a gene to its exact location within the genome and identify its nucleotide sequence and the protein that it encodes. The traditional map-based or positional cloning strategy narrows down the gene location by using genetic recombination in biparental populations that segregate for the gene of interest  Figure . This apMutagenesis and the Resistance gene Enrichment and Sequencing) (Association genetics with R gene enrichment Sequencing) (Mutgenesis Chromosome flow sorting and short-read Sequencing) (Targeted Chromosome-based Cloning via long-range Assembly)  , AgRenSeuencing) , MutChrouencing) , and TACssembly) . The comResistance gene Enrichment Sequencing (RenSeq) approach, which is an NLR gene-targeted, resistance gene enrichment and sequencing method. Solanum tuberosum). The workflow was to first construct a customized target enrichment library (bait library) comprised of a series of 120-mer biotinylated RNA oligonucleotide. These were designed based on previously annotated NLR-like sequences from potato, tomato, tobacco, and pepper genomes and aimed to fully cover each NLR-gene sequence with bait probes of at least 80% sequence identity. This bait library was then used to capture and enrich the NB-LRR genes from a genomic DNA library of potato which was then sequenced using Illumina technology. Through this approach, the number of annotated NLRs in the potato genome was increased from 438 to 755. Furthermore, they applied RenSeq successfully in identifying SNP markers that co-segregate with the resistance against late blight pathogen Phytophthora infestans in two independent segregating populations of wild Solanum species.MutRenSeq was the earliest developed TEnSeq pipeline applied to clone rust resistance genes in the wheat genome. It is based on the Solanum species, Hordeum vulgare), hexaploid wheat (T. aestivum), tetraploid wheat (T. durum), red wild einkorn (T. urartu), domesticated einkorn (T. monococcum), and three goatgrass species . The first successful application of MutRenSeq pipeline in cloning wheat rust resistance genes was the rapid cloning of wheat stem rust resistance genes Sr22 and Sr45. Later, its high efficiency was again demonstrated through the successful and rapid identification of Yr7, Yr5, and YrSP, which are the first three cloned major R genes against wheat stripe rust .Following the successful application of RenSeq on genetic mapping on the ipe rust . The notGenome-Wide Association Studies (GWAS) to permit the cloning of R genes from a host diversity panel. Sr46 and SrTA1662 from a panel Ae. tauschii accessions. They also identified the previously cloned Sr33 and Sr45 genes within this diversity panel. In this approach, the GWAS analysis was based on the use of unique K-mer (sub-sequences) markers rather than single nucleotide polymorphisms. K-mers associated with resistance phenotypes were then used to identify the candidate NLRs.The AgRenSeq pipeline also utilizes the NLR-gene capture method but integrates with Pm2 by Rph1 from barley by SuSr-D1, a suppressor gene of stem rust resistance from the wheat cultivar ‘Canthatch’ . This allowed the identification of candidate genes within the physical interval delineated by the position of markers closely linked to Lr22a.The TACCA pipeline was first described by Pgt, which is about 600 aa. The prediction of fungal effectors was facilitated in recent years by the increasing availability of fungal genome sequence data, and especially the development of approaches to assemble the two haploid genomes of these dikaryotic organisms separately in the case of rust fungi  Pgt isolates and virulent mutants. These two Pgt effectors are quite distinct from each other in sequence, but both are Haustorial Secreted Proteins (HSPs). Coincidentally, the two genes are located adjacent to each other in the Pgt genome  encounters its corresponding effector (Avr gene). Most NLRs confers race-specific resistance and are effective in all growth stages of the host, therefore, these are often termed as all stage resistance (ASR). Race non-specific resistance describes resistance that is effective against all races of a pathogen species and sometimes may also be effective against multiple pathogens. These are normally quantitative traits conferring partial resistance that is able to slow down disease development. Wheat stem rust resistance gene Sr2, stripe rust resistance gene Yr36, and three multi-pathogen resistance loci Lr34, Lr46, and Lr67, all fall into this race non-specific category (Lr34, Yr36, and Lr67 since 2009 revealed these APRs encode an ABC transporter, a kinase-START protein, and a hexose transporter, respectively. They appear to each have their own resistance mechanism, function constitutively and often increase the basal level of resistance of the host, which is different from the recognition based NLRs. An important consideration is that some race-specific genes are also only effective at the adult plant stage, such as the NLR-encoding Lr22a (Effective resistance gene stewardship requires reference to the mechanism of resistance conferred by the available genes and their ability to work in combination. Rust resistance genes may be either race-specific or non-race-specific . Race-spcategory . Most ofng Lr22a .Sr2, Lr34, and Yr36 all have been reported to have some additive effects when in combination with certain seedling resistance genes. Given the distinct resistance mechanisms between APRs and ASRs based on current knowledge, these synergistic or additive resistance phenotypes between the APR and ASR are likely the result of combining two different modes of resistance.In terms of gene stewardship, Avr genes, these new tools should lead to more rational deployment strategies to maximize resistance durability. Although the TEnSeq strategies were initially developed specifically for resistance genes, these approaches can also be applied to genes with other functions and also adapted to other crops with large and complex genomes similar to wheat (There are roughly over 200 rust resistance genes that have been officially designated in wheat. Only a handful of these has been cloned to date, while many additional effective genes are expected to exist in wheat relatives. The advances in wheat rust resistance gene cloning reviewed in this paper, in particular, the TEnSeq pipeline which includes the MutRenSeq, MutChromSeq, TACCA, and AgRenSeq, will facilitate the identification of a much broader repertoire of wheat resistance genes. This will provide many more tools for marker-assisted selection in wheat breeding as well as the raw gene sequences to pursue gene stacking via transgenic gene cassettes. Together with advances in identifying genetic variation in rust to wheat .JZ wrote the manuscript. All authors reviewed and provided edits.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Takotsubo syndrome (TTS) is a complex and still poorly recognized heart disease with a wide spectrum of possible clinical presentations. Despite its reversibility, it is associated with serious adverse in-hospital events and high complication rates during follow-up. Multimodality imaging is helpful for establishing the diagnosis, guiding therapy, and stratifying prognosis of TTS patients in both the acute and post-acute phase. Echocardiography plays a key role, particularly in the acute care setting, allowing for the assessment of left ventricular (LV) systolic and diastolic function and the identification of the typical apical-midventricular ballooning pattern, as well as the circumferential pattern of wall motion abnormalities. It is also useful in the early detection of complications  and monitoring of systolic function recovery. Left ventriculography allows the evaluation of LV function and morphology, identifying the typical TTS patterns when echocardiography is not available or wall motion abnormalities cannot be properly assessed with ultrasound. Cardiac magnetic resonance provides a more comprehensive depiction of cardiac morphology and function and tissue characterization and offers additional value to other imaging modalities for differential diagnosis . Coronary computed tomography angiography has a substantial role in the diagnostic workup of patients with acute chest pain and a doubtful TTS diagnosis to rule out other medical conditions. It can be considered as a non-invasive appropriate alternative to coronary angiography in several clinical scenarios. Although the role of nuclear imaging in TTS has not yet been well established, the combination of perfusion and metabolic imaging may provide useful information on myocardial function in both the acute and post-acutephase.The online version of this article (10.1007/s12574-020-00480-y) contains supplementary material, which is available to authorized users. Takotsubo syndrome (TTS), also known as takotsubo cardiomyopathy, stress-induced cardiomyopathy, or apical ballooning syndrome, is an acute and transient heart failure syndrome originally reported by Dr Sato in 1991 in a Japanese textbook and by Pavin et al. in Europe in 1997 . Chest pRecently, a classification of TTS according to the type of triggering event has been proposed Table  4].Tab.Tab4].Electrocardiographic abnormalities (ST-T elevation in the majority of cases) resembling those detectable in acute coronary syndromes are common, as well as increased biomarkers reflecting myocyte damage despite unobstructed coronary arteries. For this reason, TTS has been classified as ‘myocardial injury’ in the last universal definition of myocardial infarction and categorized as myocardial infarction with non-obstructive coronary arteries (MINOCA) . The patOwing to the wide spectrum of clinical presentations, diagnosis of TTS is often challenging. However, early recognition of this syndrome is key to adopting an appropriate therapy. Multimodality imaging is helpful in reinforcing the clinical suspicion of TTS in the early phase, allowing confirmation of the diagnosis, even retrospectively, after ruling out other clinical entities that should be considered in the differential diagnosis. The aim of this consensus document is to discuss and review the utility of multimodality imaging, including left ventriculography, echocardiography, computed tomography (CT), magnetic resonance imaging, and nuclear imaging, in the diagnostic workup of TTS.Several diagnostic criteria for TTS have been proposed, including those issued by the Mayo Clinic, the Japanese guidelines , the TakAlthough initial studies suggested that prognosis of TTS is benign  , 14, recCoronary arteries are generally normal or near-normal in TTS. Although echocardiography is the first-line imaging modality in patients with suspicion of TTS, assessment of coronary anatomy is crucial in TTS diagnostic workup to rule out alternative diagnoses. As recommended by current guidelines on acute coronary syndromes (ACS), cardiac catheterization should be performed in all patients with acute cardiac ischaemia and ST-segment elevation or in patients without ST-segment elevation according to the individual risk profile, especially for patients with a low-intermediate probability of TTS , 17–21.Coronary anatomy should be carefully evaluated using multiple angiographic views, ensuring that all lesions have been assessed in at least two orthogonal projections. Obstructive atherosclerotic plaques can be observed in about 1 in 10 of TTS patients , 18. It The diagnosis of TTS can also be established during cardiac catheterization through LV opacification. Biplane left ventriculography in the right and left anterior oblique projections allows for the assessment of nine LV segments and differentiation of TTS from anterior ST-elevation myocardial infarction (STEMI) in the majority of cases , 22. TheAlthough not specific for TTS, coronary anatomical variants, including myocardial bridging or long and tortuous arteries, are common findings on CA . LikewisKey pointsCA should be performed in ACS according to guidelines in all STEMI and intermediate to very high-risk non-STEMI presentations.Obstructive coronary stenosis does not represent an absolute exclusion criteria for TTS, but if coronary artery disease is present, it should not be sufficient to explain the observed regional WMAs.Biplane left ventriculography allows assessment of LV function and morphology in TTS.In selected cases, intravascular imaging and functional testing to rule out vasospasm may be helpful for the differential diagnosis.Transthoracic echocardiography (TTE) is the first-line imaging modality for the evaluation of patients with TTS . If suspFirstly, TTE may help to detect the distribution of WMAs and determine the morphologic anatomical variant , 34.Videos S1 and S2) .In clinical practice, the most important application of contrast echocardiography is LV cavity opacification for the detection of WMAs  Fig. 1010 and thOf note, myocardial contrast echocardiography performed within 24 h after TTS onset revealed reversible perfusion defects in involved segments, which improved faster than WMAs .3D echocardiography is a useful method to quantify chamber measurements with higher accuracy and reproducibility than 2D echocardiography  Figs. 11 and 12.These ‘advanced’ echocardiography techniques, including strain imaging, coronary flow assessment, contrast echocardiography, and 3D echocardiography, are emerging and hold promise in improving our understanding of the pathophysiology underlying TTS. However, it should be acknowledged that the clinical utility of these techniques has not been fully established and physicians should not make clinical decisions solely on this basis.Key pointsLV mechanics are profoundly and transiently impaired in TTS, in a pattern that differs from AMI.Whilst EF and WMAs resolve in TTS, some subtle wall strain abnormalities can persist.The coronary microcirculation is transiently compromised in TTS, but its physiological role is still unclear.Contrast echocardiography should be used in patients with poor acoustic window to better define WMAs and the presence of intraventricular thrombus.Compared to 2D echocardiography, 3D echocardiography enables more accurate assessment of regional WMAs and may be a useful tool to better detect and assess RV involvement, even in the acute care setting.As an additional prognostic tool, RV strain could be useful in assessing the extent of RV involvement.Although echocardiography and left ventriculography have mainly been used to diagnose LV WMAs in patients with TTS, CMR plays an important role in the comprehensive assessment of the functional and structural changes that occur in patients with clinical suspicion of TTS . CMR repVideos S7–S10). These planes enable precise visualization of the contractile dysfunction pattern  including short-axis plane covering the entire left ventricle and three long-axis planes , should be systematically performed during CMR  , 97.Recently, CMR-feature tracking (FT) has been introduced as a new method to quantify regional LV deformation similarly to speckle tracking echocardiography, providing a more reliable and accurate assessment of (dys-)synchrony as well as rotational parameters . In TTS CMR has the advantage of enabling multiparametric myocardial tissue characterization. Specifically, CMR can rule out ischaemic myocardial injury or myocarditis using late gadolinium-enhancement (LGE) imaging Fig.  96]. Wh. Wh96]. Although patchy areas of LGE can be detected in TTS patients using low SI thresholds, the transient nature of this myocardial injury, which generally disappears within few days or weeks, as well as the absence of LGE at high SI thresholds are distinctive patterns that differentiate TTS from AMI and myocarditis. It has been suggested that an increase of extracellular matrix rich in collagen-1, as a finding of transient fibrosis, might be accountable for the LGE observed in TTS .In conclusion, these evidences suggest that the absence of LGE at high SI thresholds is still a diagnostic criterion for TTS, keeping in mind that small amounts of reversible patchy areas of LGE can be transiently detected using low SI thresholds and do not represent an absolute exclusion criterion during diagnostic work-up.The impact of LGE on clinical outcomes in TTS remains controversial. Although some authors have reported worse clinical prognosis in patients with LGE, no differences in clinical outcomes between patients with and without LGE have been found in a multicentre prospective registry , 103.T1 mapping techniques, where T1 time of injured myocardium is quantitatively and more objectively assessed, can be useful to detect very subtle areas of damage in patients with TTS Fig. . AssessmUp to date, cut-off values of native T1 mapping have not been established in this setting. This technique is still considered a research tool and its impact on clinical practice and outcome needs to be further investigated.in vivo the acuity, extent, and severity of myocardial stunning in TTS [Myocardial oedema is another distinctive feature of TTS. The exact pathophysiological mechanisms underlying the development of myocardial oedema in TTS is still unclear. Increased LV wall stress and/or transient ischaemia seem to be responsible for inflammatory injury of cell membrane, with subsequent transmembranous leakage of water and large molecules leading to extracellular oedema. It can be detected by T2 weighted black blood imaging (T2BB) or newer mapping techniques (i.e. native T1 mapping and T2 mapping). T2BB allows to recognize oedema due to the suppression of both fat and flowing blood signal. In T2BB imaging, a triple inversion recovery fast-spin echo sequence is used in contiguous short-axis views of the left ventricle. A ratio of mean SI of the myocardium compared with that of the skeletal muscle (T2 SI ratio) ≥1.9 is generally accepted to define oedema. Myocardial oedema is generally localized in segments with abnormal contraction (a-hypokinesis) showing a diffuse or transmural distribution. Its acute extent also correlates with acute release of both catecholamines and N-terminal pro-B-type natriuretic peptide . It is ag in TTS  . Additionally, CMR is recommended in case of incomplete recovery (persisting ECG changes and/or WMAs at echocardiography) and atypical presentation  to assess tissue characterization and definitively corroborate TTS diagnosis.Key pointsCMR can visualize the entire spectrum of functional and structural changes that occur in patients with TTS.typical regional WMAs ;presence of reversible tissue injury (oedema);CMR provides additional value to other imaging modalities for differential diagnosis , pathophysiological insights, and detection of complications (e.g. LV thrombi) in TTS.In the acute phase, CMR is recommended in doubtful TTS cases, especially if diagnosis of another type of MINOCA (e.g. myocarditis) requires a different therapeutic approach.In the post-acute phase, CMR is mandatory in all patients within 2 months, especially in case of persisting ECG abnormalities and/or regional WMAs at echocardiography, in order to definitively confirm the diagnosis of TTS.absence of irreversible tissue injury (LGE) when SI thresholds of 5 standard deviations are used.CMR diagnostic criteria for TTS include the combination of:Technological developments over recent years have emphasized the role of coronary computed tomography angiography (CCTA) in the evaluation of patients with suspected TTS .An advantage of both CCTA and CMR is the ability to evaluate the heart in any potential plane, overcoming the limitations imposed by suboptimal acoustic windows on echocardiography . MoreoveCCTA has been proposed as a non-invasive alternative to CA in stable and pain-free patients with no ST-segment elevation at onset and convincing clinical  and echocardiographic  findings consistent with TTS. This approach could be advantageous, particularly in patients with delayed presentation (>48 h after chest pain onset) or when CA is not available on site .Cardiac CT, beyond coronary artery disease, allows assessment of LV function and WMAs. Nowadays, cardiac CT also provides myocardial characterization with late iodine enhancement imaging comparable with that obtained from CMR .Owing to multiplanar imaging , 116 thaUp to date there is no evidence for delaying invasive CA in patients with ongoing acute chest pain and acute ECG changes. In such a clinical scenario, patients should be managed according to current guidelines for STEMI or ACS until culprit coronary artery disease has been ruled out , 21.in stable patients with low suspicion of ACS;in patients with suspected TTS recurrence;in other conditions generally associated with TTS such as critical illnesses .CCTA may be chosen instead of CA:Moreover, CCTA can be considered as a non-invasive valuable alternative in life-threatening conditions where CA is highly likely to cause complications .Key pointsThe main indication for CCTA in TTS patients relates to the assessment of the epicardial coronary arteries and the ruling out of coronary artery disease.Cardiac CT also plays an important role in the diagnostic workup of patients with acute chest pain and doubtful TTS diagnosis to exclude other conditions such as pulmonary embolism and aortic dissection.Stable patients with low suspicion of ACS;Patients with a history of TTS and suspected recurrence;Critical clinical conditions usually associated with TTS  or where CA is highly likely to cause complications .CCTA may be chosen instead of CA in some peculiar situations:Although the role of nuclear imaging in TTS has not yet been well established in clinical practice, nuclear imaging can provide assessments of myocardial perfusion and metabolic activity, improving our understanding of the pathophysiology of TTS and potentially aiding diagnosis.123I-beta-methy-iodophenyl pentadecanoic acid (123I-BMIPP) single-photon emission computed tomography (SPECT) was more severely impaired than myocardial perfusion depicted by 201thallium scintigraphy in acute TTS patients [Some studies have reported that fatty acid metabolism depicted by patients , 120. Re123I metaiodobenzyl guanidine (MIBG) scintigraphy can assess myocardial sympathetic nerve terminal activity and detect adrenal and ectopic pheochromocytoma. Even in the subacute phase, the adrenergic hyperactivity observed in TTS results in decreased uptake and increased washout of 123I-MIBG from the heart  shows abnormal glucose metabolism alongside normal myocardial perfusion in TTS. Recently, some studies have been conducted at different phases during the TTS time course [Cardiac positron emission tomography (PET) using [e course , 124. InThe pathophysiology of TTS remains challenging and controversial. Several hypotheses have been proposed to explain this unique cardiac disorder , 125–128Key pointsAlthough the role of nuclear imaging in TTS has not yet been well established in clinical practice, myocardial perfusion and sympathetic nerve innervation can be assessed by SPECT and MIBG.123I-MIBG scintigraphy) may allow diagnosis of TTS in patients with delayed presentation, even within a few months of the suspected triggering episode.In the context of normalized LV wall motion, the delayed recovery of glucose metabolism (by FDG-PET) and sympathetic innervation (by Coronavirus disease 2019 (COVID-19) is a tremendous infectious disease firstly recognized in Wuhan, Hubei, China, that has spread throughout to other provinces in China and impetuously involved worldwide many countries causing a pandemic and a public health crisis of global proportions in 2020. Cardiovascular involvement, including acute coronary syndromes, myopericarditis, and pulmonary embolism, has been associated to the clinical course of a substantial proportion of patients affected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Myocardial injury emerged as an important predictor of worst outcome. Shi et al. report a prevalence of 19.7% of cardiac injury, defined by high blood levels of high-sensitivity troponin I, among 416 patients with confirmed diagnosis of SARS-CoV-2 . In thisAs with other coronaviruses, an intense systemic inflammatory response secondary to SARS-CoV-2 may produce demand ischaemia and/or possible coronary plaque disruption. Moreover, the detection of increased levels of multiple cytokines and chemokines in patients hospitalized in intensive care unit, suggests a direct myocardial damage as an alternative potential mechanism .Noteworthy septic status, hypoxaemia, and metabolic acidosis; complications often detected in SARS-CoV-2 patients, are established triggers for secondary TTS.Owing to the detection of myocardial inflammatory infiltrates documented in TTS patients, possible shared pathophysiological pathways in occurrence of myocardial dysfunction might be hypothesized in patients with SARS-CoV2 experiencing myocardial injury . TTS andImaging diagnostic tests are not routinely used in the emergency context of pandemic disease, the European Association of Cardiovascular Imaging recommend performing echocardiography at least in patients with laboratory or electrocardiographic signs of cardiac injury . The susAlthough the current challenging clinical scenario makes serial invasive and not invasive diagnostic testing difficult, whenever possible multimodality imaging should be performed in patients with SARS-CoV-2 and cardiac injury to distinguish type II myocardial infarction, myocarditis, and TTS, and to adopt the most appropriate therapeutic strategy.TTS is a complex clinical entity, with a wide spectrum of clinical presentations and varying levels of risk and haemodynamic stability, which requires tailored management strategies depending on the different scenarios. Multimodality imaging plays a key role in establishing the diagnosis, guiding therapy and stratifying prognosis in both the acute and post-acute phases of TTS Figs. ; Table 5Supplementary file1 (AVI 2875 kb)Supplementary file2 (AVI 2992 kb)Supplementary file3 (AVI 11181 kb)Supplementary file4 (MP4 1033 kb)Supplementary file5 (MP4 1028 kb)Supplementary file6 (MP4 1231 kb)Supplementary file7 (AVI 2389 kb)Supplementary file8 (AVI 18591 kb)Supplementary file9 (AVI 2065 kb)Supplementary file10 (AVI 2053 kb)Below is the link to the electronic supplementary material.


Dengue fever is one of the most common viral infections affecting humans. It is an expanding public health problem, particularly in tropical and subtropical regions. No effective vaccine or antiviral therapies against Dengue virus (DENV) infection are available. Therefore, there is a strong need to develop safe and effective therapeutic strategies that can reduce the burden and duration of hospitalizations due to this life-threatening disease. Oligonucleotide-based strategies are considered as an attractive means of inhibiting viral replication since oligonucleotides can be designed to interact with any viral RNA, provided its sequence is known. The resultant targeted destruction of viral RNA interferes with viral replication without inducing any adverse effects on cellular processes. In this review, we elaborate the ribozymes, RNA interference, CRISPR, aptamer and morpholino strategies for the inhibition of DENV replication and discuss the challenges involved in utilizing such approaches. Dengue virus (DENV) is an important mosquito-borne viral pathogen known to cause dengue and severe dengue. An annual incidence of approximately 300 million DENV infections has been estimated and the disease burden is significantly higher in tropical and subtropical regions  1]. It . It 1]. Drug repurposing approaches are also being attempted against DENV. Previously approved drugs such as chloroquine, celgosivir, ribavirin, prednisolone, lovastatin underwent clinical trials, but none of these showed promising activity against dengue ,19. InhiDespite intense research efforts, finding an effective antiviral agent is elusive and an effective strategy for treating dengue is urgently needed. Targeting the viral proteins for antiviral activity is the major convention used for identifying antiviral agents. Although, targeting the viral genome to inhibit replication of the virus is an attractive strategy which can be employed against different viruses. Oligonucleotides are generally employed to target the viral genomes, resulting in inhibition of replication and or translation, also known as silencing of the viral genome expression. In this review, we discuss the different oligonucleotide-based approaches employed/available for targeting dengue.These nucleic acid-based agents generally interact with a specific region of the target genome and downregulate the gene expression or completely disrupt its normal expression. Oligonucleotide-based approaches use different types of RNA or DNA molecules such as ribozyme, siRNA, miRNA, CRISPR, aptamer and morpholino to silence the expression of the viral genome.Ribozymes are small catalytic RNA molecules which are targeted towards RNA for cleaving at a particular site . In 1982Hammerhead and group-I intron ribozymes are the most promising in inhibiting viral replication due to their strong binding efficacy and catalytic activity. The hammerhead ribozymes (hhRz) have a length of 40–50 nucleotides forming two domains; a functional domain and a structural domain A. The stThe Group I intron ribozymes have recently been in focus for therapeutic development due to their strong catalytic activity to cleave either single stranded or homologouslypaired double stranded RNA at a defined base . The groAedes aegypti tRNAval promoter for suppressing DENV-2 replication in lentivirus-transduced C6/36 mosquito cells. These hhRz also contained 3′polyA tail of 6o bases length which helped in cleaving the target RNA within the secondary structures. These hhRz RNA cleaved the targets at NUH triplet site. In C6/36 cells transduced with pantropic retroviral vectors expressing certain hhRz RNA, 100-fold suppression of DENV-2 replication was observed. Carter et al. [Aedes aegypti cell lines stably expressing these group I trans-splicing introns, suppression of DENV-2 virion production was observed compared to non-transformed cell lines. Though anti DENV group I introns can suppress the virus replication in mosquito cells, this alone is not sufficient to prevent the emergence of escape mutants of DENV. Later, the research group coupled the splicing activity of group I intron with death-upon-infection strategy [Nawtaisong et al.  designedr et al.  designedstrategy . In thisstrategy . Caenorhabditis elegans embryo development. Small interfering RNA (siRNA) and microRNA (miRNA) are the two main categories of small RNAs which are being investigated extensively for their multiple roles in regulating gene expression and antimicrobial activity. siRNAs and miRNAs have emerged as practically modular and adaptable therapeutics for treating viral infections [RNA interference is an evolutionary mechanism of gene regulation that is induced by small RNA molecules along with a specific endonuclease. In the year 1990, Napoli and Jorgensen  first refections .Both siRNA and miRNA exert gene silencing in a post transcriptional manner . siRNA cIn the siRNA-mediated RNAi pathway, dsRNA is processed into 21–30 nt-long short siRNA molecules that act as a module of silencing mechanism. The silencing pathway involves chopping of dsRNA into siRNA by a RNase III family of enzyme, known as “Dicer” and introduce two nucleotide overhangs at the 3′ ends. Dicer has four distinct domains: an RNase/Helicase domain, dual catalytic RNase III domain, a double stranded RNA binding domain and a PAZ domain . Short sZhang et al.  first reThe RNA interference mediated by siRNAs is now being widely used to knock down several important viral genes in a sequence-specific manner. A study reported the designing of four siRNAs targeting DENV-1 viral membrane glycoprotein precursor (prM) gene . The stuAedes albopictus has been shown to interact with the E glycoprotein of DENV and influence its expression. Inhibition of miR-252 in C6/36 cell line led to an increased expression of DENV-2 E protein and DENV-2 RNA copies while, over expression of miR-252 reduced DENV-2 RNA copies and the expression of viral E protein [Apart from siRNA, few studies have reported the inhibition of DENV by using miRNAs. miR-252 from  protein . Another protein . A signi protein . Later,  protein  designedThough siRNAs can effectively inhibit the replication of DENV in both in vitro and in vivo, to date, there have been no in vivo studies regarding the antiviral activity of miRNA against DENV.Aptamers are a class of oligonucleotides (either DNA or RNA) which can bind specifically with a protein by three-dimensional conformation. Aptamers can be considered as an alternative to antibodies. In 1990, Jack Szostack demonstrated the isolation of a short RNA oligonucleotides which were able to interact with organic dyes and coined the term “Aptamer” . In the Aptamers are short DNA/RNA sequences which can fold into a complex three-dimensional structure and bind towards a targeted strand A. The inThe library is generated for providing different confirmation variabilities of oligonucleotides towards a target sequence. In the second step, the oligonucleotide pool is incubated with the target molecule and selected based on interaction ability. The unbound oligonucleotides are removed from the reaction setup, as those are not able to interact and contain less binding capability. In the final step, the recovered aptamer or oligonucleotides are PCR amplified to generate a pool of high affinity aptamers with less variability. Maximum rounds of SELEX are generally performed to obtain an aptamer population with the highest binding affinity towards the target. The aptamers are flexible and can be further modified to improve their physical and chemical appearance such as stability, nucleophile attack escape, and others .N-7-5′ capping. The aptamers can be promising therapeutic agents due to high efficacy, low toxicity, strong pharmacokinetics mechanism and low cost.The antibody-dependent enhancement (ADE) mechanism is a special immunological phenomenon which is generally associated with secondary DENV infections. During secondary infection with a heterologous serotype, non-neutralizing, cross reactive antibodies are produced against the prM protein and these antibodies enhances the infection of DENV by binding to Fc receptors on the monocytes/macrophages. This phenomenon is known as ADE and is considered as one of the major contributors to disease severity . Due to TM (Eteplirsen) has already been approved by the U.S. Food and Drug Administration for the treatment of the genetic disorder, Duchenne muscular dystrophy (DMD) [Morpholinos are a synthetic antisense oligonucleotide which can bind to a target mRNA by Watson-Crick base pairing and block the translation through RNase H-independent steric blockade. In 1985, Summerton came up with a morpholino structure which showed strong pharmaceutical properties and therapeutics applications . The morhy (DMD) . In receThe morpholinos are non-ionic in nature and possesses a backbone composed of morpholine rings and phosphonodiamidite inter-subunit linkages. The most common type of morpholino possessing a phosphorodiamidate backbone is known as phosphorodiamidate morpholino (PMO). The PMO A was synOnce morpholino is inside the cell, it covers the mRNA target sequence like masking tape. The typical translation machinery starts with the binding of small ribosomal subunit within the 5′ UTR region and starts the formation of the initiation complex B. The stKinney et al.  first de4-fold when compared with untreated cells at a concentration of 4 µM. This group also reported the inhibition of DENV in monocyte-derived dendritic cells (MDDCs) by using vivo-MO-1 targeting 3′SL of DENV [Vivo morpholino has also been reported to be a potent antiviral against DENV which can bind to the RNA and can penetrate directly into the cells . The fir of DENV .Clustered regularly interspaced short palindromic repeats (CRISPR)-based antiviral technology is also currently in focus to treat DENV infection. During the last few years, CRISPR technology has introduced itself as an effective antiviral therapeutic strategy against several viral pathogens. The Type II and VI CRISPR/Cas system, especially for Cas 9 and Cas 13, is the most extensively characterized system which has been reported to exert strong antiviral activity.N-sulfotransferase 1 (NDST1), exostosin glycosyl transferase 1 (EXT1) and endoplasmic reticular membrane complex (EMC), and oligosaccharyltransferase have been identified to be important in different stages of the DENV life cycle through CRISPR-based genetic screens [The life cycle of flaviviruses is complex and relies on host factors. CRISPR-based genetic screens have been used to identify the target proteins that are essential for DENV replication. SPCS 1 proteins, AXL receptor tyrosine kinase (AXL), RAB5C, RABGEF, N-deacetylase and  screens ,72,73,74 screens . A CRISP screens .Properties of different oligonucleotides based antiviral approaches against DENV have been summarized in O-methyl-ribose–1,5-anhydrohexitol nucleic acid (MeORNA–HNA) aptamer targeting the rat vascular endothelial growth factor 164, and a threose nucleic acid (TNA) aptamer targeting human thrombin have also been reported [Theoretically, oligonucleotides can be designed and targeted towards any gene of interest, which makes them an efficient tool for a broad range of applications. Although oligonucleotide approaches have advantages, they have faced many hurdles during clinical applications. Localized and inefficient delivery of oligonucleotides is the major drawback noticed for all the approaches. Due to their uncharged nature, oligonucleotides such as morpholino face poor cellular uptake and rapid clearance . Interesreported ,121. SucThe first licensed DENV vaccine Dengvaxia® led to considerable controversy worldwide due to major limitations . It is c


Tineola bisselliella provides an intriguing example of this phenomenon. The caterpillars of this species have adapted to feed on keratin-rich diets such as feathers and wool, which cannot be digested by most other animals and are resistant to common digestive enzymes. Inspired by the hypothesis that this ability may be conferred by symbiotic microbes, we utilized a simple assay to detect keratinase activity and a method to screen gut bacteria for candidate enzymes, which were isolated from feather-fed larvae. The isolation of DNA from keratin-degrading bacterial strains followed by de novo genome sequencing resulted in the identification of a novel bacterial strain related to Bacillus sp. FDAARGOS_235. Genome annotation identified 20 genes with keratinase domains. Proteomic analysis of the culture supernatant from this gut bacterium grown in non-nutrient buffer supplemented with feathers revealed several candidate enzymes potentially responsible for keratin degradation, including a thiol-disulfide oxidoreductase and multiple proteases. Our results suggest that the unusual diet of T. bisselliella larvae promotes their association with keratinolytic microorganisms and that the ability of larvae to feed on keratin can at least partially be attributed to bacteria that produce a cocktail of keratin-degrading enzymes.The evolutionary success of insects is promoted by their association with beneficial microbes that enable the utilization of unusual diets. The synanthropic clothing moth  Tineola bisselliella is a global synanthropic pest because the caterpillars can feed on clothes and carpets made of wool [T. bisselliella has adapted to feed predominantly on materials rich in keratin, such as feathers, woolen clothes, and carpets. Keratin is particularly resistant to proteolytic degradation because of its abundant disulfide bonds (cysteine content = 7–13%), which distinguishes it from other structural proteins such as collagen and elastin [T. bisselliella larvae identified serine-like and chymotrypsin-like proteases as candidate keratinases, but neither cysteine-like proteases nor metalloproteinases were detected [T. bisselliella.In terms of species numbers, insects are the most diverse group of organisms on earth. Their evolutionary success has in part been attributed to their ability to manage associated beneficial microbes in order to utilize unusual diets . Insect- of wool . Autecol of wool . T. biss elastin . Only so elastin . A subtrdetected . To dateT. bisselliella. To determine whether the ability of T. bisselliella larvae to utilize keratin is assisted by its gut microbiome, we isolated bacteria from the guts of caterpillars fed on a diet of feathers as a sole nutrient source. The culture supernatants were screened for keratinase activity using a simple photometric assay and the most promising strains were analyzed in more detail. Here, we report the identification of novel Bacillus strains found in the T. bisselliella larval gut with the apparent ability to digest keratin. Cultivation of these strains on sterilized feathers followed by proteomic analysis of the culture supernatant resulted in the identification of complex mixtures of enzymes and other proteins that could contribute to the digestion of keratin by T. bisselliella caterpillars.Given that many insects are associated with beneficial microbes providing enzymes which are essential for the adaptation to unusual diets, we postulated that microbes could play a similar role in T. bisselliella were obtained from the Federal Institute for Materials Research and Testing  and were reared on an exclusive diet of feathers at room temperature. The feathers were surface sterilized with 80% ethanol to kill attached bacteria before feeding. Caterpillars were surface sterilized before dissection and the extraction of gut bacteria, which was achieved by macerating the entire gut in sterile phosphate-buffered saline (PBS) and preparing serial dilutions of the homogenate. Samples were taken from different regions of the gut. Isolates were tested for general proteolytic activity on casein plates, and positive isolates were cultivated with a keratin azure substrate (blue-colored sheep wool) in PBS (37 °C) to allow the photometric (A 595 nm) quantification of keratinase activity, which releases a blue dye into the supernatant  using technology of a Pacific Biosciences Sequel instrument. A total of 90,570 PacBio raw reads were assembled with HGAP3   in “all”For the detection of genes involved in keratin degradation, UniProt  was searched for the keyword “keratinase” and the resulting FASTA  and associated text files were downloaded. Non-keratinase entries  were manually identified and removed before further analysis. Pfam  domains Extracellular bacterial proteins contained in feather-supplemented media were concentrated using the Vivaspin™ ultrafiltration spin columns  according to the manufacturers’ recommendations. Protein mixtures were subsequently separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 4–12% Criterion XT gradient gels  with XT MES running buffer at 125 V for 1.5 h. The molecular weights (kDa) of the separated proteins were estimated using pre-stained markers and unstained high-mass-precision protein markers. For LC-MS/MS analysis, protein bands were excised from the gel as 10 molecular weight blocks per lane, followed by tryptic digestion as described by . LC-MS sT. bisselliella larval gut were identified by following the scheme shown in T. bisselliella larvae feeding on an exclusive keratin-based diet and cultivated serially diluted gut homogenates on casein to identify colonies with extracellular proteolytic activity. Individual colonies were then inoculated into the substrate keratin azure, which produces an easily detected blue dye when digested (Bacillus candidates (Bac18) for further genome analysis, proteomics, and keratinase assays.Keratin-degrading bacteria and candidate keratinases in the digested . We alsoThe final draft genome of Bac18 is represented by one circular chromosomal genome with a size of 5.2 Mb and additional five small contigs with a total length of 827 kb . We idenBacillus sp. FDAARGOS_235 share 83.93% of their genomes (conserved DNA) with an overall average nucleotide identity (ANI) of 99.56%. In this regard it has to be noted that the small contigs of Bac18 make up ~14% of the length of the genome. A visualization of the structural similarities of the Bac18 chromosomal sequence with Bacillus sp. FDAARGOS_235 is shown in We found that Bac18 and We searched the Bac18 genome for potential keratinase-encoding genes and identified 20 sequences with predicted keratinase domains as found in Pfam. The amino acid sequences of the 20 candidates were used as BLAST queries against keratinase entries in UniProt. We identified 63 different proteins in the feather-supplemented culture supernatant that matched sequences predicted in the Bac18 genome using our proteomics cut-off criteria . We subsT. bisselliella feed predominantly on keratin-based materials such as feathers, hairs, and wool. The midgut of these insects is anaerobic with a negative redox potential, which was proposed to facilitate the solubilization of keratin by reducing disulfide bonds, enabling digestion by more widespread proteases [T. bisselliella gut transcriptome [Caterpillars of the clothing moth roteases . Howeverroteases . Analysiroteases ,20. Althcriptome , their iTherefore, it remains to be determined whether serine proteases encoded by the host insect genome can process keratin, or whether additional enzymatic or physiological functions are required to initiate the degradation of this molecule by reducing the abundant disulfide bonds. T. bisselliella larvae [Clostridiales, Lactobacillales, and the genus Bacillus, similar to the bacteria associated with the brown house moth Hofmannophila pseudospretella, another keratin-feeding lepidopteran [Bacillus strain with a close relationship to Bacillus sp. FDAARGOS_235 and B. thuringiensis. Interestingly, Bacillus species are predominant among bacteria displaying keratinase activity, and are known to encode for several keratin-degrading proteases.An alternative explanation for the adaptation of the clothing moth to keratin-based diets is that keratinase activity is conferred by specific bacterial symbionts in the gut. Although previous studies found no abundant microorganisms in a larvae ,22, we wdopteran . After sT. bisselliella larvae promotes their association with keratinolytic microorganisms and that keratin digestion is potentially supported by bacteria in the larval gut. The thiol-disulfide oxidoreductase was particularly interesting because this enzyme can hydrolyze disulfide bonds, thus enabling the complete proteolytic degradation of keratin and subsequent nitrogen assimilation [On a keratin diet, larvae of the clothing moth develop very slowly, possibly owing to the limited availability of an easily accessible nitrogen source. Likewise, in our assays using non-nutrient buffer supplemented with whole feathers, the inoculated gut-associated bacteria showed slow growth. When clothing moth larvae or their gut-associated bacteria were provided solely with keratin (whole feathers in our experiments) as a nitrogen source, we hypothesized that nitrogen assimilation mechanisms are induced, including the expression and secretion of keratin-active enzymes ,25,26, wBacillus species encode multiple proteases representing different functional classes and different protein masses that nevertheless possess keratinase activity [We detected a surprisingly complex mixture of numerous extracellular proteases as well as enzymes that modify bacterial cell walls, peptide-binding proteins, transporters, and stress-response factors. These functions are commensurate with the stress anticipated when bacteria are forced to grow on keratin as a sole nutrient source. Previous studies of bacterial keratinases reviewed in ,19 have activity ,28.T. bisselliella also produces its own enzymes that contribute to keratin digestion, but such native keratinases have yet to be identified [T. bisselliella to occupy a unique ecological niche. Strikingly, the tarantula Chilobrachys guangxiensis, which requires powerful enzymes to digest the immobilized preys down into a liquid taken up with straw-like mouthparts into the intestines, was also found to harbor feather-degrading bacteria in the gut producing keratinases [Based on the proteins detected in supernatants of Bac18 cultivated in nutrient-free medium supplemented with feathers, we propose that keratin digestion is a multistep process beginning with the cleavage of disulfide bonds by a thiol-disulfide oxidoreductase, enhancing the accessibility of the reduced keratin polypeptide for a cocktail of exopeptidases (aminopeptidases and dipeptidyl peptidases) ,28 and eentified . The isoatinases . Such keatinases .


We report the neuropathological findings of a patient who died from complications of COVID-19. The decedent was initially hospitalized for surgical management of underlying coronary artery disease. He developed post-operative complications and was evaluated with chest imaging studies. The chest computed tomography (CT) imaging results were indicative of COVID-19 and he was subsequently tested for SARS-CoV-2, which was positive. His condition worsened and he died after more than 2 weeks of hospitalization and aggressive treatment. The autopsy revealed a range of neuropathological lesions, with features resembling both vascular and demyelinating etiologies. Hemorrhagic white matter lesions were present throughout the cerebral hemispheres with surrounding axonal injury and macrophages. The subcortical white matter had scattered clusters of macrophages, a range of associated axonal injury, and a perivascular acute disseminated encephalomyelitis (ADEM)-like appearance. Additional white matter lesions included focal microscopic areas of necrosis with central loss of white matter and marked axonal injury. Rare neocortical organizing microscopic infarcts were also identified. Imaging and clinical reports have demonstrated central nervous system complications in patients’ with COVID-19, but there is a gap in our understanding of the neuropathology. The lesions described in this case provide insight into the potential parainfectious processes affecting COVID-19 patients, which may direct clinical management and ongoing research into the disease. The clinical course of the patient also illustrates that during prolonged hospitalizations neurological complications of COVID may develop, which are particularly difficult to evaluate and appreciate in the critically ill. The coronavirus disease of 2019 (COVID-19) has spread around the world, infecting millions of individuals , 12. TypBoth direct infection, as well as parainfectious mechanisms, may contribute to a spectrum of neuropathological manifestations associated with viral infections. It is well recognized that viral infections may cause demyelinating disease in humans, including such notable examples, as subacute sclerosing panencephalitis from the measles virus and progressive multifocal leukoencephalopathy from the JC virus . CoronavThe decedent was a 71-year-old semi-retired medical professional previously diagnosed with ischemic heart disease due to coronary artery atherosclerosis. He was initially admitted to the hospital for fatigue and exertional dyspnea, consistent with his known heart disease. Coronary angiogram demonstrated mid-left anterior descending artery (LAD) obstruction (70%) along with first obtuse marginal artery (1OM) obstruction (70%). In March 2020, he underwent an elective double coronary artery bypass graft surgery. Six days after the procedure, he developed a left-sided pleural effusion and thoracentesis yielded 450 milliliters of serosanguinous fluid. His respiratory status steadily deteriorated, with worsening symptoms and increasing oxygen demand. Chest radiographs revealed patchy bilateral interstitial abnormalities, with the computed tomography (CT) highlighting parenchymal consolidation and surrounding ground-glass opacities following a peribronchovascular distribution. Nine days after his elective procedure, COVID-19 was suspected, and he subsequently tested positive for SARS CoV-2.Eleven days after his procedure, the decedent required intubation, sedation/paralysis, and intermittent prone positioning for the management of continued acute respiratory failure. One day prior to his confirmed COVID-19 diagnosis, he developed acute kidney injury (AKI), likely secondary to shock state, respiratory failure, and IV contrast administration. Continuous renal replacement therapy was required for 5 days after AKI diagnosis. He remained critically ill over the following days with a highly inflammatory state, as evidenced by rising levels of CRP, IL-6, and ferritin. Extensive supportive therapies were pursued, including vasopressor support, and stress dose steroids. Despite the initial improvement in gas exchange and lung mechanics, prognosis remained poor, and the family decided on DNR status. The immediate cause of death was attributed to medical treatment-refractory junctional bradyarrhythmia.The postmortem gross examination of the brain revealed mild brain swelling and hemorrhagic lesions disseminated throughout cerebral hemispheric white matter, ranging in size from 1 mm to 1 cm Figs. a, b. RouThe histologic examination revealed a second type of pathological lesion that on H&E was characterized by subtle areas of subcortical white matter pallor with a variable perivenular distribution Figs. a. A CD68A few microscopic cortical organizing/organized infarcts were also identified that were not appreciable on gross examination Fig. . GFAP imOur understanding of SARS CoV-2 infections and COVID-19 is rapidly evolving; significant gaps in our knowledge remain. The neuropathology in the case presented raises questions about the pathophysiology of the infection in the central nervous system (CNS) and provides insights into the neurological complications of these patients. Parainfectious conditions are well known to develop after viral infections, including SARS. In this case, the pathological lesions are distinct and the interpretation is not confounded by underlying severe global hypoxic–ischemic brain injury with concomitant regional infarcts. The striking features of the pathology presented include the range of subcortical white matter pathology, the absence of deep grey nuclei or brainstem pathology, and the lack of typical features of viral and post-viral encephalitides.We describe several types of pathological lesions that may contribute to neurological manifestations of COVID-19 patients. The widespread hemorrhagic white matter lesions identified have some of the characteristic features of AHLE. Similar hemorrhagic lesions have previously been described with SARS-CoV, but are reported within a background of extensive neuronal necrosis , which wThe fundamental question is whether the observed neuropathological lesions are a result of primary vascular disease with secondary white matter injury  or is due to a parainfectious primary demyelinating type of disease or a combination. Parainfectious conditions, such as post-viral autoimmune disorders, have been described in association with a variety of viruses, including coronaviruses , and variety of potential routes of CNS infection have been proposed . The potParainfectious neuropathological processes typically present after a latent period following an infectious illness. The prolonged course of this patient suggests that during his hospitalization, he developed a secondary disease initiated by SARS CoV-2, which raises the clinical question of when, or if, to consider evolving neurological processes during a prolonged period of intubation. Further complicating the clinical management of these patients is the microscopic white matter lesions, and cortical infarcts would not be readily apparent with routine imaging studies. Furthermore, these patients may not be stable, or appropriate, for transporting for more complex imaging studies. Our understanding of the parainfectious neuropathology of SARS CoV-2 continues to evolve, but remains a cause of either devastating outcome or morbid sequelae in many patients that do survive.


Takotsubo syndrome is an acute cardiac emergency characterized by transient left ventricular systolic dysfunction typically following a stressful event. Despite its rapidly rising incidence, its pathophysiology remains poorly understood. Furthermore, it may pass unrecognized especially if timely diagnostic imaging is not performed. Defective myocardial calcium homeostasis is a central cause of contractile dysfunction and has not been explored in takotsubo syndrome. We aimed to investigate myocardial calcium handling using manganese-enhanced magnetic resonance imaging during the acute and recovery phases of takotsubo syndrome.Twenty patients with takotsubo syndrome  and 20 age, sex and cardiovascular risk factor matched volunteers  were recruited from the Edinburgh Heart Centre between March 2020 and October 2021. Patients underwent gadolinium and manganese-enhanced magnetic resonance imaging during index hospitalization with repeat manganese-enhanced magnetic resonance imaging performed after at least 3 months.2, P<0.001) and, in affected myocardial segments, elevated native T1  and T2  values at their index presentation. During manganese-enhanced imaging, kinetic modelling demonstrated a substantial reduction in myocardial manganese uptake  consistent with markedly abnormal myocardial calcium handling. Following recovery, left ejection fraction, left ventricular mass and T2 values were comparable to matched control volunteers. Despite this, native and post-manganese T1 and myocardial manganese uptake remained abnormal compared to matched control volunteers .Compared to matched control volunteers, patients had a reduced left ventricular ejection fraction , increased left ventricular mass (86±11 versus 57±14 g/mIn patients with takotsubo syndrome, there is a profound perturbation of myocardial manganese uptake which is most marked in the acute phase but persists for at least 3 months despite apparent restoration of normal left ventricular ejection fraction and resolution of myocardial edema, suggesting abnormal myocardial calcium handling may be implicated in the pathophysiology of takotsubo syndrome. Manganese-enhanced magnetic resonance imaging has major potential to assist in the diagnosis, characterization, and risk stratification of patients with takotsubo syndrome. Moreover, it can be challenging to recognize due to its phenotypical similarities with acute myocardial infarction and is often considered when invasive coronary angiography fails to identify major obstructive coronary artery disease.4 However, the characteristic left ventricular abnormalities can be brief, and opportunities to document them with diagnostic imaging are often missed.5Takotsubo syndrome is an acute cardiac emergency that is often triggered by a stressful event and is characterized by transient and profound left ventricular systolic dysfunction, typically due to marked ‘ballooning’ of the left ventricular apex.6 However, there is a need to develop more sensitive and discriminatory imaging techniques that are less time sensitive and more specific to takotsubo syndrome.Cardiac magnetic resonance imaging is an essential diagnostic tool in the assessment of takotsubo syndrome. In addition to visualizing the hallmark regional wall motion abnormalities, it can identify complications, such as left ventricular outflow tract obstruction, mitral regurgitation, pericardial effusion and left ventricular thrombus. It is particularly useful in establishing the diagnosis of acute cardiac conditions of uncertain origin, such as myocardial infarction with non-obstructive coronary arteries and myocarditis. Previous cardiac magnetic resonance imaging studies have demonstrated the presence of acute myocardial edema in patients with takotsubo syndrome as well as some reports of persistent subtle cardiac abnormalities following recovery of normal left ventricular ejection fraction.7 Manganese-enhanced magnetic resonance imaging thus represents a promising novel approach to the assessment of myocardial calcium handling. We have previously demonstrated differences in manganese uptake in patients with dilated, hypertrophic or ischemic cardiomyopathy, suggesting abnormal myocardial calcium handling.9 However, there have been no studies assessing whether manganese-enhanced magnetic resonance imaging can detect alterations in myocardial calcium handling in takotsubo syndrome and whether this recovers during convalescence. The objectives of this proof-of-concept study were to evaluate the ability of manganese-enhanced magnetic resonance imaging to detect altered myocardial calcium handling in patients with takotsubo syndrome during both the acute presentation and following apparent recovery.Manganese, a calcium ion analogue, has paramagnetic properties and can cross intact cell membranes via voltage-gated calcium channels, providing intracellular contrast of viable tissue on magnetic resonance imaging. In 1981, Hunter et al proposed that uptake of free manganese ions in the myocardium can be used to measure calcium uptake because manganese is retained intracellularly.This was a single center case-control observational longitudinal cohort study (NCT04623788) which was conducted in accordance with the Declaration of Helsinki, the favourable ethical opinion of the Southeast Scotland Research Ethics Committee 2 (20/SS/0001) and with the written informed consent from all participants. The data, analytic methods, and study materials will be made available to other researchers on reasonable request.10 and the Heart Failure Association Takotsubo Syndrome Taskforce of the European Society of Cardiology criteria.11 This comprises of new electrocardiographic (ECG) changes , the presence of transient left ventricular dysfunction presenting as apical ballooning or focal mid-ventricular/basal wall motion abnormalities, and the absence of obstructive coronary artery disease or acute plaque rupture. Patients usually have an emotional or physical stressful trigger. We specifically excluded patients with pheochromocytoma, myocarditis or a primary isolated diagnosis of acute myocardial infarction.Adult patients (≥18 years of age) with takotsubo syndrome were recruited from the Edinburgh Heart Centre between March 2020 and October 2021. Diagnosis of takotsubo syndrome was based on the Mayo clinicControl volunteers were selected consecutively and were matched to patients with takotsubo syndrome 1:1, for age, sex and cardiovascular risk factor profile, such as hypertension, known coronary artery disease, hypercholesterolemia and diabetes mellitus. All participants were scanned at the University of Edinburgh between September 2020 and August 2021.2), New York Heart Association class IV heart failure, and women of child-bearing potential without a negative pregnancy test. Subjects receiving a calcium channel antagonist had their medication withheld 48 hours prior to manganese-enhanced imaging.Exclusion criteria for all participants were any contraindication to magnetic resonance imaging, contraindications to manganese dipyridoxyl diphosphate administration , ongoing calcium channel antagonist or digoxin therapy, renal failure  using a 30-channel anterior body matrix coil and elements of a posterior spine matrix coil. Images were acquired during expiratory breath hold with ECG gating. Cine imaging was acquired with standard steady-state free precession sequences in long and short-axis orientations as described previously.wo scans .T1 mapping was performed prospectively with modified Look-Locker inversion (MyoMaps) recovery 3). Quantitative estimation of T1 was performed in full short-axis stack from mitral valve annulus to apex and standard long-axis slices, with additional slices positioned appropriately to characterize pathology.T2 mapping was performed with T2 Myomaps  was 360×288 mm adjusted for patient body habitus as required . This was acquired in long and short-axis orientation covering the entire left ventricle.Late-gadolinium enhancement images were acquired following intravenous administration of gadobutrol  using a single breath held phase-sensitive inversion recovery short-axis stack, and long axis orientations . A standardized inversion time of 400 ms was used and adjusted as required for optimal myocardial nulling. Post-contrast T1 mapping was performed prospectively with short-axis modified Look-Locker inversion recovery stack 10 min after contrast administration.Manganese-enhanced magnetic resonance imaging was achieved using intravenous infusion of manganese dipyridoxyl diphosphate  T1 mapping was performed pre-contrast with full short-axis modified Look-Locker inversion recovery stack as above. For patients, a single short-axis slice of the diseased myocardium was identified by the supervising cardiologist and guided by native T1, T2 maps and cine images . Single short-axis T1 mapping was then performed at this slice location every 2.5 min for 30 min after commencing contrast infusion. At 30 min, a full short-axis T1 stack was repeated . For con13 Image analysis was conducted blind to patient’s details  and manganese-enhanced images were analysed separately from late-gadolinium images. Endocardial and epicardial borders were manually defined on all conventional short-axis images for volumetric and wall motion measurements and were then copied to corresponding T1 map images for analysis with minimal manual adjustments. The left ventricular basal short axis slice was identified as the image containing at least 50% of circumferential myocardium at end diastole. Papillary muscles were included in the mass and excluded from volumetric analysis.All analyses of T1, T2 maps, late-gadolinium enhancement and cine-derived volumetric and functional sequences were performed using Circle CVI  as previously validated and described in animal and human models.After contouring, an additional epicardial and endocardial offset of 20% was applied automatically to minimize partial volume effect for all T1 map analyses. In patients, T1 and T2 measurements were taken from septal segments in both pathological  and remote regions . In control volunteers, T1 and T2 measurements were made in the septal wall of the midventricular slice . Global 15Manganese uptake was calculated for a selected single short-axis slice of the diseased myocardium which was chosen based on the presence of regional wall motion abnormality, and native T1 and T2 maps. To quantify the change in T1 over time, regions of interest were drawn as described above. For serial T1 imaging post manganese, manually drawn regions of interest from the pre-contrast image were transferred to all subsequent post-contrast images to ensure consistency. The rate of myocardial manganese uptake (Ki) was determined by Patlak modelling as described previously.t-tests and Wilcoxon signed rank and unpaired t-test or Mann-Whitney tests where appropriate. Sensitivity analyses were performed excluding the focal subtype of takotsubo syndrome or the presence of dual pathology. Correlations were assessed using linear regression analysis. Statistical significance was taken as two-sided p<0.05.All statistical analysis was performed with GraphPad Prism . Categorical baseline variables were presented as number (%) and compared using Chi-squared test. Continuous data were assessed for normality using the D’Agostino-Pearson test and presented as mean±standard deviation or median [interquartile interval]. Cardiac function, myocardial manganese uptake, volumetric assessment and parametric mapping values were compared using paired Student’s Twenty-five patients with takotsubo syndrome were recruited into the study although five patients were withdrawn: 3 were unable to complete the cardiac magnetic resonance scan due to claustrophobia and 2 had an infarct pattern of late-gadolinium enhancement on magnetic resonance imaging and an isolated primary diagnosis of acute myocardial infarction . The patSixty infusions of manganese dipyridoxyl diphosphate were completed during the course of the study (mean duration of 10 min). There were no changes in the electrocardiogram, heart rate or blood pressure  followinElectrocardiography demonstrated either ST-segment deviation or T-wave changes in all patients, with evidence of QT interval prolongation in some patients. All patients had a degree of left ventricular impairment on baseline echocardiographic imaging. All patients underwent invasive coronary angiography, which, demonstrated normal coronary arteries in half of the population, with the remaining having non-obstructive coronary artery disease . Two patMost patients underwent cardiac magnetic resonance imaging within a median of 4 days (range 1 to 18 days) of symptom onset. Compared to matched control volunteers, patients with takotsubo syndrome had reduced left ventricular ejection fraction , increasCompared to matched control volunteers, patients with takotsubo syndrome had elevated native T1 and T2 values in both the pathological and remote myocardial segments . FollowiOne patient underwent cardiac magnetic resonance imaging 18 days after the onset of presentation symptoms, and there was complete resolution of regional wall motion abnormalities and normal left ventricular systolic function. Despite this, manganese-enhanced T1 mapping demonstrated a pattern of reduced myocardial manganese uptake consistent with apical takotsubo syndrome .All 20 patients returned for follow-up and underwent repeat manganese-enhanced magnetic resonance imaging 100 days  after symptom onset  with 11 2= 0.6 and 0.7 respectively, In patients with takotsubo syndrome, native and post-manganese T1 values were reduced compared to those obtained after the acute index event but remained higher than the matched control volunteers (P=0.02 and P=0.003 respectively). T2 values in the previously pathological myocardium normalized and were comparable to matched control volunteers (P=0.12). Left ventricular mass and left ventricular wall thickness  were also reduced and correlated with the reduction in myocardial native T2 values  and sarcolipin, were markedly altered suggesting that this may be responsible for the associated ventricular dysfunction. Using myocardial manganese uptake as a measure of the flux of intracellular calcium ions, we confirm that marked alterations in myocardial calcium handling appear to play a major pathophysiological role in takotsubo syndrome, especially in the acute high-risk period. Indeed, such a mechanism could explain why levosimendan can improve cardiogenic shock during acute severe cases of takotsubo syndrome since it augments myocardial calcium binding to troponin C. Although this is an important fundamental mechanistic observation, we cannot determine whether these alterations are a consequence or a cause of the takotsubo syndrome and this will require further study.For the first time, we have demonstrated a profound Takotsubo syndrome is partly defined by the apparent dramatic improvement of left ventricular function following the acute dramatic emergent presentation. The rapid transition from severe left ventricular systolic dysfunction and low ejection fraction to apparently normal left ventricular function and ejection fraction within days is a characteristic feature of this condition. However, this can create challenges for the diagnosis of this condition since unless the index of suspicion is high and the diagnosis is considered early, these typical features will resolve and the opportunity to undertake diagnostic imaging may have passed. In this regard, manganese-enhanced magnetic resonance imaging may provide an opportunity to identify the typical distribution of myocardial abnormalities despite normalization of regional wall motion abnormalities. This provides a unique opportunity to diagnose this condition at later time points and help resolve potential diagnostic uncertainties especially where the clinical suspicion of the diagnosis was initially low. There can also be major diagnostic uncertainty in cases of dual pathology where acute myocardial infarction triggers a takotsubo syndrome as demonstrated by two examples in our case series. Manganese-enhanced magnetic resonance imaging could therefore prove invaluable in identifying and discriminating cases of takotsubo syndrome, especially when the precipitating events is caused by another primary cardiac condition.21 The majority of patients continue to complain of fatigue, tiredness and reduced exercise tolerance despite the presence of a normal left ventricular ejection fraction. Continued long-term impairment in cardiac energetic status and reduced exercise maximal oxygen capacity has previously been described and are likely to be linked to the abnormalities of myocardial calcium handling.21 Indeed, 60% of our cohort described ongoing symptoms at follow up, compatible with a heart failure-like syndrome with a substantial impact on quality of life.There are major implications for the protracted abnormalities in myocardial calcium handling. First, these findings could account for the persistence of symptoms and reduced exercise capacity reported by patients who have apparently recovered from takotsubo syndrome.25 Indeed, long-term rates of cerebrovascular and cardiac events and recurrent takotsubo syndrome approach 10% and 2% per year.24 We have recently reported abnormal myocardial manganese uptake in patients with dilated or hypertrophic cardiomyopathy as well as those with acute myocardial infarction.9 Interestingly, levels of myocardial manganese uptake were more impaired in patients who had recovered from takotsubo syndrome than those with dilated cardiomyopathy despite the latter having more marked left ventricular systolic dysfunction.9 Indeed, such was the profound suppression of manganese enhancement during the acute phase of takotsubo syndrome that it was comparable to the infarcted myocardium of patients with acute myocardial infarction and the densely fibrotic regions in the left ventricle of patients with hypertrophic cardiomyopathy.In contrast to the prior belief that the heart recovers spontaneously and completely without clinical sequelae, patients with takotsubo syndrome are now recognized to have substantial long-term morbidity and mortality which is comparable to that of acute myocardial infarction.21Whilst it would have been ideal to assess patients with takotsubo syndrome prior to their incident event, the longer-term persistence of abnormal myocardial calcium handling does suggest an underlying cardiomyopathy which is only brought to light following an acute stressful event. As such, we may never see normalization of myocardial calcium handling, and is again in keeping with previous studies describing a heart failure-like phenotype in this patient cohort.There are potential opportunities for manganese-enhanced magnetic resonance imaging to play an important role in prognostication and the assessment of novel treatment interventions. Persistent perturbation of myocardial calcium-handling may identify those patients who are at risk of incident or recurrent cases of takotsubo syndrome. Whilst this would seem intuitive, large prospective patient cohort studies are required to establish whether this is indeed the case. In addition, there are currently no proven treatments to improve the symptoms and clinical outcomes of patients with takotsubo syndrome. The assessment of myocardial calcium handling using manganese-enhanced magnetic resonance imaging could provide a very useful surrogate biomarker of treatment efficacy. This is particularly important since standard measures of cardiac function, such as left ventricular ejection fraction, appears to be normal in patients who have recovered from takotsubo syndrome, and this has limited the field in terms of assessing the efficacy of potential preventative therapeutic interventions. It is noteworthy that persistent abnormalities of myocardial calcium handling were present despite the initiation of angiotensin converting enzyme inhibitor or beta-blocker therapies in many of our patients.27 Second, our study population size was modest, although we detected substantial and large abnormalities in myocardial calcium handling. Future multicenter studies of larger patient populations are needed to demonstrate the robustness, generalizability and clinical utility of our findings in this proof-of-concept study. Third, the Patlak model assumes that perfusion is normal. Whilst we excluded epicardial coronary flow disturbance on invasive angiography in all patients, microvascular impairment and reduced myocardial perfusion may have contributed to reductions in manganese uptake. However, although microvascular impairment is a potential feature in takotsubo syndrome,28 this abnormality improves as myocardial function normalizes29 and we observed persistent long-term abnormalities of myocardial manganese uptake following apparent recovery. This suggests that microvascular impairment alone is unlikely to be the cause of the marked myocardial manganese abnormality seen in our takotsubo syndrome cohort. Finally, there are no currently available preparations of manganese-based contrast medium for widespread clinical use. However, this is likely to change with commercially available preparations anticipated in the near future.Our study has several limitations that should be acknowledged. First, there have been prior concerns regarding toxicity of unchelated forms of manganese. However, the chelated form of manganese used here retains the necessary properties for intracellular myocardial manganese uptake without any demonstrable adverse hemodynamic or arrhythmic effects.In conclusion, we have conducted the first proof-of-concept study of manganese-enhanced magnetic resonance imaging in patients with takotsubo syndrome. Using kinetic modelling, we have observed dysfunctional myocardial calcium influx in patients with takotsubo syndrome which is most striking during the acute episode but persists despite improvement of edema and myocardial function. We believe that manganese-enhanced magnetic resonance imaging holds major promise for the diagnosis, risk stratification and monitoring of disease, with the potential for the assessment of novel treatment interventions.Supplemental Publication Material