Danaus plexippus), we factorially manipulated dietary macronutrient availability and exposure to zinc, a common metal contaminant in urban habitats that can be toxic but also has nutritional properties. We tested whether (1) the ability to survive zinc exposure depends on dietary macronutrient availability and (2) whether individuals exposed to elevated zinc levels display higher expression of antioxidant genes, given the roles of antioxidants in combatting metal‐induced oxidative stress. Exposure to elevated zinc reduced survival only for monarchs developing on a low‐macronutrient diet. However, for monarchs developing on a high‐macronutrient diet, elevated zinc exposure tended to increase survival. In addition, monarchs exposed to elevated zinc displayed higher expression of antioxidant genes when developing on the low‐macronutrient diet but lower expression when developing on the high‐macronutrient diet. Altogether, our study shows that organismal survival and oxidative stress responses to anthropogenic zinc contamination depend on the availability of macronutrient resources in the developmental environment. In addition, our results suggest the hypothesis that whether zinc acts as a toxicant or a nutrient may depend on macronutrient supply. Environ Toxicol Chem 2022;41:1286–1296. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.Biologists seek to understand why organisms vary in their abilities to tolerate anthropogenic contaminants, such as heavy metals. However, few studies have considered how tolerance may be affected by condition‐moderating factors such as dietary resource availability. For instance, the availability of crucial limiting macronutrients, such as nitrogen and phosphorous, can vary across space and time either naturally or due to anthropogenic nutrient inputs . Organisms developing in more macronutrient‐rich environments should be of higher overall condition, displaying a greater ability to tolerate metal contaminants. In monarch butterflies ( Anthropogenic activities expose natural populations to a range of chemical contaminants, including pesticides, pharmaceuticals, and heavy metals  generate oxidative stress by removing electrons from water molecules, producing reactive oxygen species (ROS) such as superoxide radicals, hydroxyl radicals, and hydrogen peroxide  to test how variation in dietary macronutrient availability influences heavy metal tolerance. Heavy metals are a persistent threat to insect pollinators such as monarchs, because metal contamination can occur in host plant leaf tissue and nectar in polluted areas  provided in floral wicks. We avoided collecting milkweed plants along roadsides. At eclosion, we transferred butterflies to large mating cages (24 × 24 × 36″) for 5–7 days prior to collecting eggs for the experiment . Butterflies in mating and egg collection cages had ad libitum access to 10% honey water provided in sponges. After mating, individual females were transferred to egg‐laying cages, each containing a single A. syriaca plant in a water wick and a moist towel to maintain humidity. We replaced plants daily. Each day, we transferred plants containing eggs to 32‐oz. plastic cups and stored the cups in a climate chamber . Eggs used for the experiment were collected from the parental females over 1 week in July 2019.All experimental monarch larvae originated from eggs produced by more than 10 wild‐collected females reared in common garden conditions. These parental individuals were originally collected as eggs or early instar larvae from the University of Minnesota Saint Paul (USA) campus over a 3‐week period in June 2019. To start a population for our rearing experiment, we reared wild‐caught individuals in the greenhouse in cages (60 × 60 × 60 cm) with ad libitum access to campus‐collected milkweed stalks . On average, the zinc concentration of the elevated zinc diet was approximately 5× higher than the control diet . Our elevated zinc treatment was approximately in the range of zinc concentrations observed in plants along highways , and the low‐macronutrient diet contained approximately 25% less nitrogen than the high‐macronutrient diet . The average carbon‐to‐nitrogen ratio of the low‐macronutrient diet was 7.731 , and for the high‐macronutrient diet it was 5.885 . We did not manipulate the milkweed powder portion of the diet, because this serves as a feeding stimulus rather than a nutrient source.We reared monarch caterpillars on a semi‐artificial diet designed for monarchs by O.R Taylor at Monarch Watch, validated in previous studies . As expected, monarchs that developed on the elevated zinc diet tended to have higher thorax zinc concentrations, and monarchs developing on the high‐macronutrient diet tended to have higher thorax phosphorous concentrations , we transferred larvae with a paintbrush from milkweed to one of the four treatments (40 larvae/treatment). Diet was provided in 16‐oz. plastic cups (2 larvae/cup). Throughout larval and pupal development, we housed all individuals in a climate chamber maintained at 25 °C with a 15:9‐h light:dark photoperiod. We positioned all cups on their sides to provide adequate space for butterfly eclosion. To confirm variation in monarch nutrient accumulation across treatments, thorax concentrations of zinc and phosphorous (in mg/kg) were measured in 2) of five randomly selected mature eggs from each dissected female. All dissections were performed using a Leica M165C microscope at ×10 magnification while the butterfly abdomen was submerged in a 1× phosphate‐buffered saline buffer; eggs removed from the ovaries consistently lay on their sides in these conditions, facilitating an area measurement despite the three‐dimensional bullet shape of the egg.We calculated monarch development time as the time between the day larvae were transferred to semi‐artificial diet treatment and the day of eclosion. Monarch survival was quantified as whether an individual survived to eclosion. After eclosion, we labeled all butterflies on the hindwing with a fine‐tipped black sharpie. All females that eclosed with undamaged wings were transferred to a large cage (61 × 61 × 61 cm) in the greenhouse for an egg development period of 7 days before being stored in a −20 °C freezer until ovary dissections. We measured egg production as the number of fully yoked and chlorinated eggs in the ovaries of dissected females. We also measured the average egg area  analyses. First, we selected actin because it is one of the most frequently used reference genes for qPCR in insects and has been shown to be the most stably expressed reference gene across different developmental stages in a broad range of insect taxa , zinc exposure , or their interaction  on caterpillar body mass prior to dissection. We dissected fat body tissue because this organ plays a central role in insect metabolic functions that are highly sensitive to variation in nutrient supply . We extracted RNA from fat body tissue dissected from larvae at the fifth instar (17 days after transfer to each dietary treatment). Although there was no way to account for potential differences in development time across individuals at the time of dissection, analysis of variance indicated that there were no significant effects of macronutrient availability  for complementary DNA synthesis using Invitrogen SuperScript II reverse transcriptase and quantitative real‐time (qRT‐)PCR. All PCR primers were developed and validated at UMGC; all qRT‐PCR was performed using an Applied Biosystems 7900HT real‐time PCR instrument. Two technical qPCR replicates were produced for each monarch RNA sample.For statistical analyses, we used R Studio Ver 3.6.3 . No effects of zinc exposure  or an interaction between macronutrient availability and zinc exposure  on egg production were detected. In addition, we detected no effects of macronutrient availability , zinc exposure , or an interaction between macronutrient availability and zinc exposure  on average size of eggs produced by adult females. Across all females, forewing length was not significantly correlated with either egg number  or egg area , so it was not included as a covariate in either analysis.There was a significant effect of macronutrient availability on egg production , had adult forewing lengths that were approximately 4% shorter , and had approximately 15% slower growth rates , on average. There were no effects of zinc exposure or an interaction between macronutrient availability and zinc exposure on development time, forewing length, or growth rate , monarchs in the low‐macronutrient/control zinc treatment tended to have lower antioxidant gene expression Figure , and expConsistent with our initial hypothesis that tolerance to heavy metal contamination is macronutrient dependent, we found that elevated zinc exposure negatively impacted survival only of monarchs developing on a low‐macronutrient diet Figure . Given tIn nature, resource dependence may help explain why individuals vary in their abilities to tolerate pollutants across spatial scales. For instance, nutrient availability may vary greatly across landscapes, either naturally or due to differential nutrient inputs from human activities  compared with monarchs on the control treatment Table . This isIt is unclear why a given level of zinc exposure might have harmful effects in individuals with low access to macronutrient resources but potential nutritional and regulatory benefits in individuals with high access to macronutrient resources. One hypothesis is that more nutrient‐limited individuals have fewer energetic resources available to invest in physiological machinery required to efficiently regulate internal zinc levels. For instance, key regulators of zinc homeostasis are the metallothionein proteins, which sequester zinc, enable its antioxidant properties, and play a central role in detoxification  are not also considered (Sih et al., https://doi.org/10.1002/etc.5305.The Supporting Information is available on the Wiley Online Library at Alexander M. Shephard: Conceptualization; methodology; formal analysis; funding acquisition; investigation; visualization; writing—original draft. Emilie C. Snell‐Rood: Conceptualization; funding acquisition; methodology; writing—editorial advice. Noah S. Brown: Investigation; writing—editorial advice.This article includes online‐only Supporting information.Figure S1.Click here for additional data file.Supplementary information.Click here for additional data file.Supplementary information.Click here for additional data file.


Acute aortic syndromes are life-threatening conditions with high morbidity and mortality. The principal pathological feature is acute wall damage with possible evolution towards aortic rupture. Accurate and timely diagnosis is mandatory to avoid catastrophic consequences. Indeed, misdiagnosis with other conditions mimicking acute aortic syndromes is associated with premature death. In this view, cardiovascular imaging is necessary for the correct diagnosis and management. Echocardiography, computed tomography, magnetic resonance imaging, and aortography allow for diagnosis, guarantee immediate treatment, and detect associated complications. Multimodality imaging is essential in the diagnostic work-up to confirm or rule out acute aortic syndromes. The aim of this review is to highlight the contemporary evidence on the role of single cardiovascular imaging techniques and multimodality imaging in the diagnosis and management of acute aortic syndromes. Acute aortic syndromes (AAS) represent a spectrum of interrelated disorders characterized by the disruption of the aortic integrity, and are associated with high morbidity and mortality. These conditions include aortic dissection (AD), accounting for the majority of AAS (80%), intramural hematoma IMH, ~15%), and penetrating aortic ulcer  5%, and p,3.AD is described as a separation of the aortic wall layers caused by an intimo-medial tear, resulting in the creation of a false lumen that propagates within the medial layer. Thus, AD typically shows the appearance of a dissection flap, an entry tear, and two aortic channels . No modern classifications have substituted the older Stanford and DeBakey ones that remain the most commonly used. AD involving the ascending aorta is defined as a Stanford type A dissection, regardless of distal extension; when the disruption is distal to the left subclavian origin, on the contrary, it is considered to be type B. The DeBakey classification recognizes types I, II, and, III, with type I affecting both the ascending and descending aorta, type II the ascending aorta and the arch only, and type III involving the descending aorta but sparing the arch and the ascending aorta. The Stanford classification is used to guide different treatment options. Indeed, surgery is recommended in type A AD, while medical therapy is suggested in uncomplicated type B AD. IMH is instead a haematoma created within the media layer and secondary to the rupture of the vasa vasorum. Finally, PAU is an ulceration of an atherosclerotic aortic plaque penetrating towards the internal elastic lamina and into the media .The advent of multi-modality imaging in the assessment and management of AAS has multiple applications, not only for diagnosis, but also for risk stratification and planning of the acute intervention, as well as for the evaluation of potential complications and/or the candidacy for intervention or re-intervention in the subacute or chronic phases .In this document, our aim is to critically review the distinctive multi-modality imaging features of AAS, while at the same time providing an update and overview of diagnostic strategies and the current management of these disorders.Echocardiography is renowned for being a non-invasive, safe, and accurate imaging modality used in the clinical evaluation of cardiovascular disease and plays an important role in the diagnosis of aortic diseases. Transthoracic echocardiography (TTE) can be performed at the patient’s bedside, as well as in the emergency and critical care units, and for this reason the European Society of Cardiology guidelines  recommenThe diagnosis of AD by TTE is based on detecting intimal flaps in the aorta. The sensitivity and specificity of TTE range from 77–80% and 93–96%, respectively, for ascending AD, and varies according to the use of contrast agents . Since sTOE is currently considered as one of the reference techniques in AD diagnosis, with a very high diagnostic accuracy for the detection of both ascending and descending aortic diseases B 12]. I. I12]. IThanks to its high spatial resolution, full assessment of thoracoabdominal aorta, short acquisition time, and wide availability, CT represents the ideal technique for diagnosing AAS.The typical scan protocol first includes a non-contrast CT followed by an arterial acquisition . A late The sensitivity and specificity of CT for AD is approximately 98–100% , and it The classical finding of AD in CT is an intimal flap with a partition between the true and false lumen. Supporting signs are the internal displacement of intimal calcifications, the delayed enhancement of the false lumen, the widening of the aorta, mediastinal, pleural, or pericardial hematoma , and sig2 when gadolinium is used, and the poor assessment of arterial wall calcification.Magnetic resonance imaging (MRI) is a highly accurate, noninvasive imaging modality, with a sensitivity and specificity of almost 98% for detecting AD . AdditioIn a suspected case of AD, the standard MRI examination should begin with rapid spin-echo black blood acquisitions covering the aorta to outline aortic shape and diameter and to rule out alterations in wall structure . In the In the past, aortography represented the gold standard for the diagnosis of AD . In moreAortography visualizes all features of AD: the intimal flap, true lumen , false lumen, craniocaudal extension, indirect visualization of the coronary arteries, possible aortic regurgitation, and aortic rupture E. VariouIMH is classified among AAS and represents about 15% of cases. Specifically, it is defined as Type A when it affects the ascending aorta and the aortic arch and type B when it involves the descending aorta. . IMH is Non-contrast CT from the supraortic vessels to the aortic carrefour is crucial to diagnosing IMH. IMH is visible on unenhanced CT typically as an eccentric, hyperdense (60–70 HU), crescentic area of thickening of the aortic wall (>0.5 cm), showing no enhancement after administration of intravenous contrast that extends in a longitudinal, non-spiral fashion A,B. The Other CT findings that can be useful to predict the negative evolution of the IMH include an IMH high mean IMH thickness greater than 10–11 mm, compression of the true aortic lumen, a maximal ascending aortic diameter greater than 50 mm, or descending aorta greater than 45 mm, association with PAU, and the presence of pericardial effusion . These hMRI has the unique ability to determine the age of the hematoma based upon T1 and T2 signal characteristics ,48,49. DBreath-hold ECG-gated fast spin echo images acquired with T1 and T2 weighting are used to assess aortic calibre, aortic wall thickness, and aortic wall signal change. These are acquired as contiguous transverse sections from the apexes to the infrarenal abdominal aorta and also as sections paralleling the long axis of the aortic arch. On MRI, the spin-echo black blood images of an IMH show a crescent-shaped area of eccentric thickening, and exhibit the expected signal characteristics associated with the transition of hemorrhage from deoxyhemoglobin (low T1 and T2) in the hyperacute stage to intracellular methemoglobin  in the subacute stage and extracellular methemoglobin (high T1 and T2) in the late stage of IMH A.2 represents a limitation, in addition to the difficulty in monitoring patients in the acute phase.Dynamic SSFP sequences are used to assess aortic valve function and the calibre of the aortic root. These are acquired in both transverse and coronal planes through the left ventricular outflow tract (LVOT). If aortic regurgitation is present, its severity can be quantified via flow sensitive phase contrast sequences. Finally, a gadolinium-enhanced aortic angiography study could be performed. However, the risk of nephrogenic systemic fibrosis with GFR < 30 mL/min/1.73 mA small study suggested three magnetic resonance angiography (MRA) parameters to distinguish IMH from type B dissection: (1) no visualized entry tear, (2) no contrast uptake in aortic lesion on first pass angiography, and (3) no contrast uptake in the aortic lesion on the equilibrium phase T1-weighted sequence . In addiIn the diagnosis of IMH, aortography has a more limited role compared to classic dissection because the intima is intact and, as a result, the lumen of the aorta remains unaffected . The onlTOE is among the methods for detecting PAU and evaluating possible complications B. AlthouCT today represents the most used technique for the diagnosis and follow-up of PAU thanks to its high spatial resolution, which allows for a detailed assessment of atherosclerotic plaques, calcium, and the aortic wall . CT characteristic features of PAU are an aortic out-pouching in the presence of aortic intima calcifications and severe atherosclerotic disease. The edges of the out-pouching are usually irregular or jagged  Figure C. PAU caCT imaging can be helpful in stratifying the risk of patients with PAU to select the appropriate management. In particular, patients with PAU diameters over 13–20 mm and more profound than 10 mm and concomitant IMH are associated with a worse prognosis . ChoosinPAU can be well visualized on MRI, as well as the associated IMH. Ulcer craters can be documented on spin-echo images as a signal void within the thickened aortic wall . The immThe diagnostic challenge consists in distinguishing PAU from the ulcerated atherosclerotic plaque and the IMH from the intraluminal thrombus. In 1990 Yucel et al. studied seven patients with acute chest pain and penetrating aortic ulcers by MRI imaging compared with angiography and CT. They demonstrated that MRI, thanks to the ability to image the aortic wall, was superior to angiography in depicting the extent of intramural thrombus and was superior to CT in differentiating acute IMH from atherosclerotic plaque and chronic intraluminal thrombus . As noteCompared to AD, patients with PAU are older and present risk factors for atherosclerosis such as arterial hypertension, dyslipidemia, and smoking . Once agIn an emergency scenario, a rapid comprehensive diagnostic work-up is necessary, including clinical assessment , laboratory data (D-dimer and troponin amongst others), chest X-ray, and ECG in order to expedite the appropriate aortic imaging studies ,64,65,66In this setting, bedside TTE combined with additional TOE have the priority in order to exclude new-onset aortic regurgitation, pericardial effusion, or to visualize proximal dissection. Modern ultrasound equipment is in fact mobile and particularly useful at the bedside for unstable AAS and/or for detecting painless AD . TOE evaGiven the optimal accuracy of all multi-modality imaging modalities, imaging protocols should be adapted to local expertise and availability, the patient’s clinical condition, and the target of interest .CT technology has currently replaced invasive diagnostic angiography for the thoracic and abdominal aorta. CT is rapid, accurate, and has high spatial resolution, but requires patient transportation and, thus, stable haemodynamic conditions .MRI angiography is also capable of providing high-resolution aortic imaging with three–dimensional post-processing; it needs neither iodinated contrast nor ionizing radiation, being particularly useful for patients intolerant to contrast due to allergy or renal failure ,72,73. MAfter an AAS, surviving patients require lifelong follow-up. The imaging approach is crucial in prognostic assessment and to detect evolution and complications. The cardiac imaging techniques utilized for surveillance are echocardiography, CT, and MRI. CT is the most commonly performed for the long-term follow-up after an AAS. However, especially in young patients, this technique must consider radiation exposure. MRI is a valid alternative and avoids the use of iodinated contrast in addition to radiation exposure. Furthermore, in the IMH, it identifies the evolution of temporal bleeding and new hemorrhagic episodes. Cardiac imaging follow-up is indicated both in patients managed with medical therapy alone and in those treated surgically or with TEVAR ,77,78,79A multimodality imaging approach in the AAS is indispensable for accurate and timely diagnosis. In this setting, prompt diagnosis is mandatory considering this life-threatening condition which requires adequate and immediate treatment. In addition, multimodality imaging adds key information about urgency indicators and the associated complications. A correct and high-quality diagnostic work-up improves the poor prognosis in this emergency condition. Advances in cardiovascular imaging techniques have led to a better understanding of the pathophysiology and improvement in diagnosis and management. The appropriate choice of imaging modality is based on the accuracy, advantages, and limitations of the techniques. However, the clinical conditions of the patient and local availability and expertise should be also considered. The correct application of the imaging algorithm and cardiovascular techniques is crucial for the diagnosis and management of AAS.


Despite major advances in immunosuppression, allograft rejection remains an important complication after heart transplantation, and it is associated with increased morbidity and mortality. The gold standard invasive strategy to monitor and diagnose cardiac allograft rejection, based on the pathologic evaluation of endomyocardial biopsies, suffers from many limitations including the low prevalence of rejection, sample bias, high inter-observer variability, and international working formulations based on arbitrary cut-offs that simplify the landscape of rejection. The development of innovative diagnostic and prognostic strategies—integrating conventional histology, molecular profiling of allograft biopsy, and the discovery of new tissue or circulating biomarkers—is one of the major challenges of translational medicine in solid organ transplantation, and particularly in heart transplantation. Major advances in the field of biomarkers of rejection have paved the way for a paradigm shift in the monitoring and diagnosis of cardiac allograft rejection. We review the recent developments in the field, including non-invasive biomarkers to minimize the number of protocol endomyocardial biopsies and tissue biomarkers as companion tools of pathology to refine the diagnosis of cardiac rejection. Finally, we discuss the potential role of these biomarkers to provide an integrated bio-histomolecular diagnosis of cardiac allograft rejection. Heart transplantation (HTx) remains the most valuable therapeutic option for patients with end-stage heart failure ,2,3. DesOverall, there is a strong and growing literature suggesting a crucial role of invasive and non-invasive biomarkers to better monitor, detect, and refine the diagnosis of allograft rejection after heart transplantation. In this article, we review existing data concerning the use of (i) circulating non-invasive biomarkers of allograft rejection to reduce the number of protocol EMB and (ii) tissue biomarkers as a companion tool for the pathologist to better characterize allograft rejection.During the last two decades, important resources have been allocated to the search for an accurate non-invasive biomarker of allograft rejection. These biomarkers can be classified into two categories: Those reflecting allograft injury and those reflecting the inflammatory and allo-immune processes underlying allograft rejection. Due to the potential consequences of missing and not treating acute cardiac allograft rejection, these biomarkers are required to be highly sensitive for rejection even at the cost of low specificity. Despite dozens of promising studies and candidate biomarkers, only two have been approved by the Food and Drug Administration (FDA) and applied in routine clinical practice, mostly in North America (AlloMap and donor-derived cell-free DNA [dd-cfDNA]) . The oveTroponins are structural proteins of the myocardium, expressed almost exclusively in the heart. They consist of three subunits: Troponin T, C, and I. Troponin T comprises two tissue subunits, one of which is myocardium-specific, cTnT. cTnT is a biomarker mainly used in the diagnosis of acute coronary syndromes and myocarditis. However, its elevation in the blood does not reflect the underlying mechanism, but only the existence of myocardial necrosis. Therefore, the use of this circulating marker of myocardial injury, which is a rapid, non-invasive, and routinely performed assay, has been evaluated as a potential component of the strategy for the non-invasive monitoring of cardiac allograft rejection. Multiple studies, mostly retrospective single-center studies, have reported conflicting results concerning the association between troponins and rejection ,27. TheyIn a longitudinal study including unselected HTx recipients and applying a rigorous statistical approach, the temporal evolution of Troponin T did not predict the occurrence of ACR both in the early and late course of the first year after HTx .Additionally, cTn assay should probably not be used as an isolated non-invasive biomarker of rejection since (i) only ACR ≥ 2R has been evaluated thus neglecting AMR, (ii) only severe rejections with significant allograft injury are expected to increase troponin plasma level, and (iii) no threshold has been defined (importance of time post-transplant and baseline troponin level for each patient).Healthy individuals have a small amount of cfDNA corresponding due to physiological cell death. The majority of this cfDNA is released from hematopoietic cells, with <1% release from the heart. The cfDNA is increasingly used in the medical field in oncology and pre-natal diagnosis.The dd-cfDNA, detected in the blood of transplant recipients, has been proposed as a non-invasive marker of graft injury ,30. EarlThe future developments of dd-cfDNA in heart transplantation include: (i) An ongoing multicenter randomized clinical trial , and (ii) in-depth analysis of dd-cfDNA length and the absolute number of dd-cfDNA in opposition to the relative proportion currently considered.Allomap is a non-invasive test based on gene-expression profiling (GEP) of peripheral blood mononuclear cells, that has been developed for the diagnosis of ACR in HTx. Transcripts of interest reflect diverse immunoregulatory pathways from a variety of immune and non-immune cells. After broad microarray analyses and among 252 pre-selected candidate genes, 11 were finally significantly associated with ACR . A score ranging from 0 to 40 was developed and validated. In two validation cohorts, GEP appeared to detect accurately ACR ≥ 2R on the concomitant EMB. Patients > 1 year post-transplant with scores below 30 were unlikely to have grade ≥ 2R rejection  . In the This test received approval from the FDA in 2008 and the CE marking in 2011. International guidelines support the use of GEP as a non-invasive biomarker to monitor rejection. However, the use of this screening test as an alternative to the EMB in asymptomatic patients suffers from several limitations: (i) AlloMap comprises a limited number of genes expressed by a limited number of cells compared with various pathways and cell types implicated in the pathophysiology of rejection, (ii) GEP has only been evaluated to rule out severe ACR, excluding mild episodes of ACR and AMR, (iii) its high negative predictive value to rule out ACR must be interpreted in the context of a low and declining incidence of significant ACR in HTx, (iv) GEP is not specific of rejection and its positive predictive value is low, (v) the gene signature found in the peripheral blood has never been validated in allograft biopsies, (vi) its use remains limited in Europe due to costs, limited availability, and the absence of a specific economic evaluation.Small and non-coding RNAs called miRNAs have been shown to be involved in gene expression regulation. Although miRNAs are known to be involved in many biological processes, such as development, cell proliferation, differentiation, apoptosis, and oncogenesis, increasing evidence suggests that they may play a critical role in the regulation of immune cell development and in the modulation of innate and adaptive immune responses. Several studies have reported a potential causative role of miRNAs in the pathophysiology of cardiac allograft rejection and distinct miRNA profiles in EMB from patients with or without rejection ,40,41,42In a multicenter retrospective case-control study, among 17 pre-selected candidate miRNAs, four  showed differential tissue and serum expression between rejection and normal heart allografts. There were strong correlations between tissue and serological expression of these four miRNAs. Assessment of these miRNAs in patient sera permitted very high accuracy discrimination between patients with and without allograft rejection. Since then, several observational studies, mostly case-control and single-center studies, have identified various miRNAs as non-invasive biomarkers of rejection, mostly ACR: miR-144-3p , miR-142® platform , miRNA profiling revealed that human and murine ACR share significant dysregulation of immune genes. Inflammatory miR-155 was the most differentially expressed between ACR and controls in both human and animals. Importantly, the authors of this study demonstrated that absence or pharmacological inhibition of miR-155 attenuated ACR, demonstrating the causal involvement and therapeutic potential of miRNAs.Several studies have reported on the potential interest of miRNA profiling of myocardial tissue. In a study combining human myocardial tissue (case-control) and a murine model of ACR, and performing an unsupervised analysis of miRNA .Based on large, international and deeply phenotyped cohorts, we recently identified five independent predictive variables associated with biopsy-proven AMR during the first year post-transplant and built a clinical risk prediction model for AMR. It showed excellent discrimination in the US test set and in an independent external validation cohort. Simulation analyses suggested that individualizing the EMB protocol according to the predicted probability of AMR may safely reduce the number of EMB performed. A user-friendly web-based interface allowing an open access evaluation of the pre-test probability of AMR on any EMB performed during the first year post-transplant was built  has been used to define the molecular pathways involved in cardiac allograft rejection [Microarray technology  tissue. The main advantages of using FFPE tissue are the ability to perform histopathology and the retrospective access to the blocks of inclusions, which allows longitudinal studies, crucial in heart transplantation (pre-injury diagnosis or response to treatment). Until recently, the main limitation of these techniques had been the relatively small number of targeted genes, which constrained discovery studies and the development of clinically relevant tools. As an example, our group has applied reverse transcription-multiplex ligation-dependent probe amplification (RT-MLPA), a multiplex RT-PCR technique to heart transplantation . We coul® system allows the specific capture and counting of nucleic acids  [® allows targeting up to 800 genes organized in the form of a “panel” of genes. The particularity of the nCounter® relies on the way it targets mRNA through a fluorescent “barcode” system. Each barcode is attached to a unique probe specific to a targeted mRNA, which is counted individually and directly without any amplification step. The probes need only 100 nucleotides to recognize their specific target. This sensitivity makes the nCounter® particularly efficient on samples with degraded nucleic acids and/or in low-quantity samples, such as FFPE tissue. It was recently shown in heart transplantation that a signature of 34 transcripts had a better diagnostic performance than C4d and DSA, and correlated with endothelial activation assessed by electron microscopy [The nCounterd miRNA) . The nCocroscopy .® technology in solid organ transplantation has been underlined by numerous studies [The relevance of nCounter studies . In the  studies . This ha studies ,68. ThisConfrontation with molecular data could improve the diagnosis of rejection and question the histopathological classifications of rejection. For example, as early as 2005 the Stanford team had shown that biopsies with mild or moderate ACR , which had been included in the 1R category of the 2004 revised ISHT working formulation, significantly differed from a molecular point of view . PangenoA key challenge remains to combine various sources of information in an integrated and more accurate diagnosis. Importantly, the evolution of bioinformatics techniques has contributed to the advancement in searching and predicting biomarkers, pathways, and new target drugs that allow a more precise and less invasive diagnosis .In Once the EMB is performed, on top of standard pathological evaluation, a molecular evaluation may produce valuable information, particularly when there is an uncertain diagnosis, discrepancies between the clinical presentation and pathology , or de novo DSA without pathologic features of AMR. The molecular diagnosis is seen as a companion tool and should not replace pathological evaluation. Finally, integrating pathology with molecular data and non-invasive biomarkers of graft injury may allow the analysis of the molecular activity of rejection and ongoing subclinical allograft injury. This information may be of high interest when discussing the treatment of subclinical rejection. The interest in an integrated bio-histomolecular diagnosis of rejection should be evaluated in dedicated prospective studies, both concerning the patient’s prognosis and the treatment of allograft rejection.Major advances in the field of non-invasive and tissular biomarkers of cardiac allograft rejection have paved the way for a paradigm shift in the monitoring and diagnosis of cardiac allograft rejection. An integrated bio-histomolecular diagnosis of allograft rejection combining non-invasive biomarkers of allograft injury, tissue molecular activity, and pathology may have a major impact on the management of heart transplant recipients.


Objective: The virtual reality (VR)-based path integration task shows substantial promise in predicting dementia risk. However, the reliability and validity in healthy populations need further exploration. The present study investigates the relationship between task indicators and brain structures in a healthy population using a VR-based navigation task, particularly the entorhinal cortex (EC) and hippocampus. Methods: Sixty healthy adults were randomly recruited to perform a VR-based path integration task, the digit span task (DST), and an MRI scan. The indicators of the VR-based path integration task were calculated, including the absolute distance error (ADE), degree of angle deviation (DAD), degree of path deviation (DPD), and return time (Time). The reliability of the above indicators was then estimated using the split-half method and Cronbach’s alpha. Correlation and regression analyses were then performed to examine the associations between these indicators and age, general cognitive ability (DST), and brain structural measures. Results: ADE, DAD, and DPD showed reasonable split-half reliability estimates  and nice Cronbach’s alpha estimates . All indicators correlated with age and DST. ADE and DAD were sensitive predictors of hippocampal volume, and return time was a predictor of EC thickness. Conclusion: Our findings demonstrate that the VR-based path integration task exhibits good reliability and validity in the healthy population. The task indicators are age-sensitive, can capture working memory capacity, and are closely related to the integrity of individual EC and hippocampal structures. Population aging is becoming an increasingly severe global issue. According to the United Nations Population Division, by 2050, the proportion of the elderly in the total population will reach 38% . Mild coHippocampal and entorhinal cortex (EC) dysfunction is a potential biological mechanism of cognitive aging ,5. PreviSpatial navigation function, such as PI, is hard to test using the typical paper-and-pencil neuropsychological test. Innovations in the virtual reality (VR) technique offer a promising opportunity. VR enables the creation of an ecological and immersive spatial environment for testing path integration . Most ofA recent study by Howett and colleagues has made impressive progress . They deThe present study thus investigates the reliability and validity of the VR-based triangle completion task using a healthy community sample. Split-half reliability is a convenient method to estimate the reliability when test–retest information was not available or a stubborn practice effect existed. We estimate the split-half reliability and Cronbach’s alpha for each behavior measure of PI. High-resolution MRI images are obtained for each subject, and brain structure measures, such as volumes and cortex thickness, are calculated. Our results support the reliability and validity of the VR-based path integration task. We recruited nearby community participants by distributing leaflets and posting posters from 2020 to 2022. Sixty-eight participants were recruited. After excluding those who did not complete all tests and had poor MR image quality, 60 people were enrolled (aged 24–84 years). All participants had no history of cancer or dementia, were not taking any medications that could affect cognitive abilities, and had no existing psychiatric or neurological diseases. All participants completed the demographic survey, the VR-based path integration task, the digital span task (DST), and the whole-brain MR scans. The behavior test and MRI scanning interval is no more than one week. Participants were informed of the purpose of this study and the privacy policy before data collection. The study was approved by the Ethics Committee of Hefei Cancer Hospital, Chinese Academy of Sciences. 2. When they exceeded the safe range, a red boundary warning would appear in their sight, reminding them to stop moving forward. The task was performed in an open virtual environment, and the boundary clues were projected to infinity. The natural and virtual worlds adopt a 1:1 motion ratio to eliminate vestibular mismatches and reduce nausea and other tolerance issues. The task was written in C# language using the Unity 3D engine  and compiled into an APK file running on the wireless VR headset.All participants wore a wireless VR headset  and walked in a square of 3.5 mParticipants were asked to walk an ‘L’-shaped outward path to three different locations, numbered as 1 (starting point), 2, and 3 (endpoint), flushing on the head. The number of land markers appeared sequentially, and participants walked following the guide of land markers. When reaching the third land marker, a voice will prompt participants to return to the starting location, and all the landmarks disappear. When participants believed they had reached the location, they pressed the button on the handheld controller to end the trial. Before the formal test, there are three practice trials to ensure that participants adapted to the VR environment. We set up three different scenarios in the formal test, each with unique surface details, boundary cues, and lighting. Specifically, one scene has both mountains and lawn surface features, and the other has only mountains or lawn surface features. Each scenario has nine trials. The environmental cues randomly change in each trial. Four PI indicators, absolute distance error (ADE), degree of path deviation (DPD), degree of angle deviation (DAD), and return time (Time), are calculated to evaluate the performance of participants in the spatial navigation task. ADE is defined as the Euclidean distance between the estimated and actual starting positions. DPD reflects the route distance length deviation, and DAD indicates the angle deviation between the estimated and actual positions. The measure of Time is the time taken from the third marker to the estimated position. DST is a popular neuropsychological tool to evaluate subjects’ attention and short-term memory. The test includes two parts: forward rehearsal and backward rehearsal. Forward rehearsal contains ten items, and backward rehearsal contains nine items. Each span size includes two number sequences. For each trial, the examiner read each number sequence at the rate of one number per second. The subject then repeat it loudly. Each number sequence counts as one point. The test terminates when subjects fail on two subsequent trials of a particular span-size condition. The forward score is the correct forward sequence number, and the backward score is the number of correct backward sequences. The total score equals the sum of the forward score and backward score.All participants underwent whole-brain MR structural image scans during the experiment using a 3.0 T Philips Achieve system and an 8-channel sense head coil. The 3D-T1-weighted images were obtained in the fast acquisition gradient echo sequence prepared by magnetization. Scanning parameters include voxel size = 1.0 × 1.0 × 1.0 mm3, TR = 7.0 ms, TE = 3.2 ms, matrix size = 256 × 256 pixels, slice thickness = 1.0 mm, scanning time = 4 min and 59 s.The whole brain segmentation was performed using FreeSurfer 6.0. The cortex parcellations followed the Desikan-Killiany atlas and subcortical segmentation followed the automatic subcortical atlas in FreeSurfer. We used the Desikan-Killiany atlas because it was the default cortex atlas of FreeSurfer, widely used in clinical studies. Two senior radiologists reviewed the images and manually corrected the offsets. Based on previous studies, we selected the EC and hippocampus as regions of interest (ROIs) ,20. All p < 0.05 was considered significant  to calculate Cronbach’s Alpha, which is a coefficient representing the consistency of “items” (variables or repetitions) across observations (such as participants). Split-half reliability was calculated using R language and the split-half package  using thnificant . GraphPanificant . To deteadjusted p < 0.05).p < 0.05). DPD and DST show a marginally significant association with DST (p = 0.065). p-values of which were corrected for multiple comparisons. r = −0.36, adjusted p = 0.013; r = −0.31, adjusted p = 0.038) and the right hippocampus volume . DPD was negatively related to the left hippocampus volume . Time was negatively related to the left EC average thickness . See p = 0.074; ADE and right hippocampus volume: p = 0.057; DAD and right hippocampus volume: p = 0.089; DAD and right hippocampus volume: p = 0.043; DPD and left hippocampus volume: p = 0.348; Time and left EC thickness: p = 0.013).We performed a whole-brain correlation analysis of all brain segmentations, the r = −0.32, adjusted p = 0.02; r = −0.31, adjusted p = 0.04). DAD was negatively related to the right temporal pole cortex and left supramarginal cortex thickness. DPD was negatively related to the left superior frontal cortex thickness, superior temporal cortex thickness, and insula cortex thickness , and right superior frontal cortex thickness, superior temporal cortex thickness, pars opercularis cortex thickness . Time correlated negatively with the left superior temporal cortex thickness, posterior-cingulate cortex thickness, and superior frontal cortex thickness  and right pars opercularis cortex thickness .In addition to the hippocampus and EC ROIs, other brain regions also correlated with VR pathway integration performances. ADE and DAD were negatively related to the right cerebellum-cortex volume , are alsAnother promising future research direction is to examine the relationship between spatial navigation and the sense of agency (SoA). SoA refers to the feeling of control over one’s actions. Previous studies have demonstrated the role of SoA during continuous body movements . In addiThe present study demonstrates that the primary measures of the VR-based path integration task have reasonable split-half reliability and Cronbach’s alpha estimates. These measures are sensitive to age and correlate with general cognitive ability. Moreover, brain structure analysis validates the association between path integration and hippocampal areas. Our results imply that the VR-based spatial navigation test might be a powerful tool in aging research.


Genome compaction and activity in the nucleus depend on spatiotemporal changes in the organization of chromatins in chromosomes. However, the direct imaging of the chromosome structures in the nuclei has been difficult and challenging. Herein, we directly visualized the structure of chromosomes in frozen-hydrated nuclei of budding yeast in the interphase using X-ray laser diffraction. The reconstructed projection electron density maps revealed inhomogeneous distributions of chromosomes, such as a 300 nm assembly and fibrous substructures in the elliptic-circular shaped nuclei of approximately 800 nm. In addition, from the diffraction patterns, we confirmed the absence of regular arrangements of chromosomes and chromatins with 400–20 nm spacing, and demonstrated that chromosomes were composed of self-similarly assembled substructural domains with an average radius of gyration of 58 nm and smooth surfaces. Based on these analyses, we constructed putative models to discuss the organization of 16 chromosomes, carrying DNA of 4.1 mm in 800 nm ellipsoid of the nucleus at the interphase. We anticipate the structural parameters on the fractal property of chromosomes and the experimental images to be a starting point for constructing more sophisticated 3D structural models of the nucleus. In the nuclei of eukaryotic cells, individual gene loci are believed to occupy preferential positions with respect to their chromosome territory (CT) and/or other nuclear landmarks such as the nuclear envelopes and nucleoli4. Although the spiral distribution of DNAs stored in a bacteriophage capsid was visualized by transmission electron microscopy (TEM)5, the compaction mechanism of chromatins in the eukaryotic nuclei is probably more complicated than that observed in virus particles. Although computational models proposed regarding the spatial organization of chromosomes in the nucleus after genome sequencing6, imaging studies are necessary to elucidate spatiotemporal variations in the organization and three-dimensional (3D) structures of chromosomes and chromatins in the nuclei7.The genetic information recorded in the genomic DNA is implemented in cellular activities through spatiotemporal changes in the organization of a large number of chromatins, assemblies of DNA and proteins, in chromosomes, and the patterns of gene positioning affect the transcription status of the genes8. A TEM study of chemically labeled human chromosomes reported that flexible and structurally heterogeneous chains of 5–24 nm diameter are packed together without any higher-order structures9. Imaging studies using super-resolution light microscopy proposed the presence of nucleosome clutches, which are heterogeneous groups of nucleosomes leading to the formation of chromatin in human nuclei10, a power law between the size and length of chromosomes in Drosophila nuclei11, and compact domains with a diameter of approximately 160 nm in vertebrate nuclei12. In addition, fluorescence in situ hybridization was used to illustrate the spatial organization of more than two loci13.Various imaging techniques have been applied to understand the structures and dynamics of building blocks in chromatin assemblies at the molecular level. TEM observations of isolated nucleosomes have revealed a variety of higher-order structures depending on the solution conditions, and have led to different ideas on models of nucleosome assemblies15, which provides structural information on the arrangement of chromosomes, such as long-range interactions of chromatins, at each genomic locus16 and at the resolution of nucleosome level19. Studies using the Hi-C techniques have revealed the characteristic features on higher-order folding of chromatins in chromosomes, such as a broad segregation of the genome into compartments20, and topologically associated domains (TADs) of hundreds of kilobases23. Based on the distance geometry information of the loci, the 3D distributions of chromosomes were computationally derived25.An alternative approach to visualize the organization of chromosomes is the high-throughput chromosome conformation capture (Hi-C) technique26, high-voltage cryoTEM tomography27, and focused-ion beam scanning electron microscopy28. In TEM observation, as the contrast of DNA was poor against the other components in nuclei, chromatins were difficult to be identified unambiguously in frozen-hydrated nuclei as pointed out9.Three-dimensional structures of whole cells have been studied using TEM for thin sections29, but fluorescent labels are necessary to visualize specific genomic loci. In addition, only the positions of the labeled molecules are visualized. Regarding the Hi-C technique24, the influence of formamide-crosslinking on the native chromatin organization is still unclear. In electron microscopy observation except high-voltage cryoTEM, specimens are stained by osmium reagent in the presence of acetone28 or sliced after the fixation using epoxy resin26.The techniques described above are useful for studying the structures and dynamics of chromosomes, but require physicochemical modification and/or treatment of the nuclei. Super-resolution microscopy has achieved spatial resolution higher than 100 nm33. The imaging technique is advantageous to visualize the differences in X-ray absorption of substances of cells. However, as the absorption contrast is poorer than the electron density contrast measurable in X-ray diffraction using short-wavelength X-ray, the visualization of the structural organization of chromosomes in the nuclei regarding X-ray absorption contrast may be still under progress.In contrast, X-ray imaging is a potential technique to visualize the structure of the whole nuclei without sectioning and chemical labeling. Soft X-ray imaging was applied to the structural study of frozen-hydrated eukaryotic cells34, a lens-less imaging technique, helps in the visualization of the shape, size, and internal structure of non-crystalline particles as electron density without the need of sectioning and chemical labeling, owing to the penetration power of X-rays with short wavelengths. XDI has been applied to investigate the structures of frozen-hydrated biological cells with sizes ranging from sub-micrometer to several micrometers using synchrotron X-rays35. The recent implementation of X-ray free electron laser (XFEL), which provides very intense and spatially coherent X-ray pulses at high repetition rates, enables the rapid collection of diffraction patterns from a large number of non-crystalline specimens, such as metal particles37, organelles38, and biological cells39, the sizes of which are smaller than the cross-section of the XFEL pulses. As the electron density contrast of a specimen are reconstructed from the diffraction patterns, XDI structure analysis of the nuclei may be advantageous to visualize the distribution of chromosomes with the highest electron density contrast among the substances in the nuclei.X-ray diffraction imaging (XDI)Saccharomyces cerevisiae at the interphase40 using XFEL-XDI. As the structures of chromosomes in nuclei have been investigated for various biological species of eukaryote, it is interesting whether any structural architectures in S. cerevisiae are common among the species. The budding yeast S. cerevisiae is an attractive model system to study nuclear organization and its functional relevance41. At the interphase, 16 chromosomes with different DNA composition42 45, a single microtubule and the kinetochore complex to a multiprotein complex embedded in the nuclear envelope, and the telomeres are tethered to the nuclear envelope  detectors and automatically processed using a custom-made data processing software suite48. Although a focused XFEL pulse destroys a specimen particle at the atomic level, diffraction occurs from the particle before its destruction49.In XFEL-XDI experiments, diffraction patterns of the nuclei were collected by supplying frozen-hydrated nuclei into the irradiation area of the focused XFEL pulses by scanning the specimen disk Fig. ” section‒1 in diffraction space), good visibility of the interference peaks  phase41, which had larger sizes and amounts of DNAs than those of the interphase, were contained in the specimen suspension , the highest resolution achievable by our experimental setup  formed a minor fraction and predominantly appeared in the first experiment only and were slightly inconsistent with the images observed in the other imaging techniques. The elliptic-shaped nuclei may be deformed in blotting procedure in the specimen preparation  and yielded maps of the ARs smaller than 1.4. As the selected 43 diffraction patterns yielded 17 type-α, 19 type-β and 7 type-γ maps, the shapes of which were circular or semicircular, the averaged profiles excluded structure information from elliptic shaped nuclei, which may be in slight deformation.The electron density fluctuation in the profiles of the projection maps suggested that certain substructures existed in the nuclei at the interphase Fig. C. To add−1 in diffraction space), indicating that any major and specific arrangements of chromosomes and/or chromatins were poor in the spacing of 400–20 nm. Since each diffraction pattern came from a single nucleus, the averaged profile was substantially different from the diffraction pattern of a pellet of nuclei, in which inter-nuclei interference of X-rays significantly modifies the diffraction patterns from each nucleus.The profile displayed no diffraction maxima in the resolution range of 400–20 nm  was grown at 303 K in a medium containing 1%(w/v) yeast extract , 2%(w/v) bacterial peptone , and 2%(w/v) D-glucose . When the optical density of the culture medium at 600 nm reached approximately 0.5, cells were harvested and resuspended in another medium containing 1%(w/v) yeast extract, 2%(w/v) peptone, and 2%(w/v) raffinose 58. After incubation for 16 h at 303 K, the harvested cells were suspended in the same medium and incubated further for 1.5 h at 303 K.The amount of DNA in the cultivated cells was examined using flow cytometry. Cells from 1 mL culture were fixed with 70% ethanol. After washing with a buffer containing 0.2 M Tris HCl , 20 mM ethylenediaminetetraacetic acid , and 0.1%(w/v) sodium azide  (pH 7.5), the cells were incubated for 2 h at 310 K in the presence of 2 mg/mL RNase . After exchange with phosphate-buffered saline, DNA was labeled with 4',6-diamidino-2-phenylindole (DAPI) . The fluorescence from the labeled cells was measured using CytoFLEX . As a result, we observed that 85% of the cells were in the interphase  sodium azide and 0.5 mM phenylmethylsulfonyl fluoride (PMSF)  were added to the cell culture. Zymolyase  at a concentration of 10 mg/mL was used to convert the cells into spheroplasts. After a step-gradient centrifugation of spheroplast lysate, pellets of crude nuclei were suspended in a buffer containing 18%(w/v) Ficoll , 20 mM PIPES , 5 mM MgCl2 , and 1 mM PMSF  (pH 6.5), and again centrifuged at 3000 × g for 30 min to remove unlysed cells, cell wall debris, and entrapped membranes. The average diameter of the isolated nuclei was found to be smaller than 1 μm using light microscopy. Although Ficoll was used without monovalent ions in this preparation, little shrinkage of nuclei was observed in reconstructed projection electron density maps  Ficoll may be negligible when viewing at the present spatial resolution.Nuclei were isolated according to a previously reported protocol2 silicon frame with nine 1 × 1-mm2 windows covered by a silicon–nitride (Si3N4) membrane with 100 nm thickness 46 . The relative humidity inside the chamber was maintained at > 90% by supplying moist air from a generator HUM-1 . Within a few minutes, the nuclei were adsorbed onto the PLL-decorated silicon nitride membranes. The specimen disk was transferred to a plastic Petri dish, in which the humidity was controlled at 95% with a sponge containing saturated KCl solution.A 30 μL droplet of suspension of isolated nuclei was placed on the silicon–nitride membranes in a custom-made humidity-controlled chamber2, as assessed by reference observation of fluorescence from nuclei labeled with DAPI. Finally, each specimen disk was flash-cooled using liquid ethane and stored in liquid nitrogen until further use . The average number density of nuclei remaining on the membranes was approximately 7/10 × 10 μmTEM images of the nuclei dispersed on carbon membrane were taken using a JEM-2100 electron microscope  operated at an accelerating voltage of 200 kV 62.Diffraction patterns were collected using our custom-made diffraction apparatus TAKASAGO-6, the MPCCD-Octal and MPCCD-Dual detectors at the beamline 3 of the X-ray free electron laser facility SACLA42 Fig. C. The MP47. The stage was kept at approximately 80 K during XFEL-XDI experiment. The stage was moved at a maximum speed of 50 μm/33 ms to supply frozen-hydrated nuclei into the focal spot of XFEL pulses. We used the signal from the control system of the SACLA linear accelerator to trigger both the translational motion of the stage and the acquisition of diffraction patterns by the two MPCCD detectors47.Specimen disks stored in a liquid nitrogen bath were transferred to the specimen stage inside the vacuum chamber of the diffraction apparatus without frosting and temperature increaseSITENNO48. After discarding diffraction patterns with respect to the signal-to-noise ratio at a specified resolution, patterns of the two detectors were merged by taking the beam center positions in each detector and the attenuation factor of an aluminum foil placed in front of the MPCCD-Dual detector  and displayed good visibility and centrosymmetry . The maximum resolution of a diffraction pattern was defined as the highest resolution shell, where the signal-to-noise ratios of the speckle peaks were greater than 3. In the two experiments, we obtained 1333 diffraction patterns displayed the maximum resolution beyond 25 nm (corresponding to a resolution range of 40 μm-1 in diffraction space), that was near the highest resolution achieved by the specimen-to-detector distance described above.We selected diffraction patterns, which had speckle peaks with a good signal-to-noise ratio beyond a resolution of 33 nm . In the first step, we determined the most probable support, i.e. particle shape of each nucleus. PR calculations frequently yield non-realistic maps due to the lack of the very small-angle region by the beamstop and Poisson noise in X-ray detection particularly in the high-angle region. Therefore, the most probable support was extracted from 10 groups of retrieved maps separated by K-means clustering after principal component analysis for 700 independently retrieved projection maps63. In the second step, 500 electron density maps inside the support selected in the first step were independently retrieved from the diffraction pattern trimmed at a resolution of 25 nm (corresponding to 40 μm−1 in diffraction space) using the oversampling smoothness (OSS) algorithm66.For each of the selected diffraction patterns, projection electron density maps were reconstructed using two-step phase-retrieval (PR) calculations51 is defined asi-th map. When a pair of maps yields a score of less than 0.2, they are deemed realistic in many cases39.Next, we screened the OSS retrieved 500 maps by referring to the similarity score. The similarity score between a pair of mapsAfter PR calculations for 1,333 diffraction patterns, we extracted maps for illustrating the structures of nuclei through the following two steps. First, we examined whether the frequency distribution of R-factor defined asR-factor of the selected maps was 0.18 (SI Appendix Fig. A set of structure amplitudes calculated from the realistic maps (Supplementary Information.


Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received.


Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process. Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received.


Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether the authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received.