Out of 41 nodules detected on the images, 8 lesions were contoured as high-risk gross tumour volumes (GTVs). Prior to each treatment, CBCT imaging was performed to match the skull structures with the planning CT images. Immediately after another intravenous bolus injection of the iodized contrast media at the same dose as administered for the planning CT imaging, contrast-enhanced CBCT images were acquired during volumetric-modulated arc therapy delivery, thereby providing direct verification of time-averaged tumour position during treatment. The planning target volume contours were overlaid with the in-treatment CBCT images, thereby allowing us to directly visualize the localization accuracy of each GTV when the beam delivery was completed. It was visually confirmed that each GTV was accurately localized inside each planning target volume during beam delivery.A direct visualization technique for verifying intrafractional localization accuracy of multiple brain metastases during single-isocentre volumetric-modulated arc therapy has been proposed using contrast media-assisted in-treatment cone beam CT (CBCT). Contrast-enhanced planning CT images were acquired immediately after intravenous bolus administration of iodized contrast media at a dose of 2 ml kg However, the localization accuracy may be decreased during treatment. Meanwhile, intrafractional CBCT was also clinically demonstrated to verify tumour positions during volumetric-modulated arc therapy (VMAT) for tumours of the prostate4 and lungs.6 Naturally, the next step was to combine these two techniques. In particular, when the single-isocentre VMAT is employed for the multiple brain tumours,7 localization accuracy of the tumours located distant from the isocentre may be of greater concern owing to rotational positioning errors.9Contrast-enhanced CT scan has a long history of being used for visualizing brain lesions.The purpose of this study was to propose a direct procedure for verifying the accuracy of brain tumour localization by way of intrafractional contrast-enhanced CBCT. To the authors’ knowledge, this has not been reported previously.A 59-year-old female patient had a 17-year history of breast cancer with diagnosis of multiple brain metastases. After receiving breast conserving therapy as primary treatment, the patient was diagnosed with local recurrence and multiple lung metastases 12 years later. Immunohistochemical tests were positive for oestrogen receptors, but negative for progesterone receptors and Her2-neu. Progressive disease and low adherence resulted in discontinuation of salvage treatments. After the progression of multiple brain metastases, the patient was referred to our hospital for radiation therapy. Even with multiple metastases to the skin, lung, adrenal grand and liver, her Karnofsky performance status was 90%.–1 and an injection time of 90 s. 30 s after injection of the contrast media, contrast-enhanced planning CT images were acquired by a large bore CT scanner, Aquilion LB , with an exposure of 175 mAs. Out of 41 nodules detected on the images, eight lesions were contoured as high-risk gross tumour volumes (GTVs) because seven of them had a volume larger than 1 ml and one nodule having a volume of 0.7 ml was located in proximity to the brainstem.An intravenous bolus injection of iodized contrast media  was manually administered at a dose of 2 ml kgA coplanar two-arc VMAT plan was created by Monaco treatment planning system, version 5.0 , with a prescribed dose of 30 Gy in 10 fractions to the whole brain and a total dose of 45 Gy in 10 fractions to the high-risk GTVs by simultaneous integrated boost. In principle, the margin for each tumour needs to take into account both translational and rotational localization errors. In other words, the translational error in each axis may require a spatially invariant margin, while the rotational error around each axis, especially when a six-degrees-of-freedom couch is not available, may need additional margins proportional to the distance between each tumour and the single isocentre. Our couch had only three degrees of freedom; therefore, for simplicity, two different planning target volume (PTV) margins for the eight high-risk GTVs were employed depending on the distance between each target and the isocentre: 2 mm for the targets located within 5 cm from the isocentre and 3 mm for the other targets located within 10 cm from the isocentre.A thermoplastic mask combined with a mouthpiece, Precise Bite , was employed to immobilize the patient's head. Subsequently, CBCT images were acquired by kilovoltage X-ray volume imaging, version 5.0 (Elekta), to match the skull structure with the planning CT images. A three-degrees-of-freedom couch, PreciseTable (Elekta), was adjusted according to the measured registration errors. If the rotation angle error was larger than 1°, a technologist adjusted the orientation of the patient's head accordingly and the registration was performed again.Prior to the treatment, informed consent had been obtained from the patient. After the treatment plan passed the quality assurance test, the patient was treated by an Elekta research linear accelerator with an Agility multileaf collimator (Elekta).Immediately after another intravenous bolus injection of the iodized contrast media at the same dose as administered for the planning CT imaging, contrast-enhanced intrafractional CBCT images were acquired during the first arc of the two-arc VMAT delivery, with a typical imaging dose of 25 mGy. As the administration of contrast-enhanced media in all fractions could influence kidney function severely, we acquired the contrast-enhanced CBCT in only one fraction. The intrafractional CBCT imaging functionality was provided by the X-ray volume imaging system.Immediately after the beam delivery, intrafractional target positions shown on the contrast-enhanced CBCT images and the overlaid PTV contours were visually compared to verify the accuracy of target localization.9 It is our intention to calculate in-treatment tumour localization accuracy for each metastasis by a direct three-dimensional visualization technique rather than a single estimation from a skull bone matching result. The initial experience shown here seems promising and a further study should be conducted, including investigation of the optimal contrast media dose and CBCT exposure conditions, with a relatively larger patient population.It was reported that the rotational positioning accuracy may be highly arguable for single-shot stereotactic radiosurgery because a significantly large rotational positioning error may arise as a statistical event during treatmentA direct visualization technique for verifying intrafractional localization accuracy of multiple brain metastases during single-isocentre VMAT has been proposed using contrast media-assisted in-treatment CBCT. The PTV contours were overlaid with the in-treatment CBCT images, thereby allowing us to directly visualize each GTV in reference to each PTV position when the beam delivery was completed. It was confirmed that each GTV was accurately localized inside each PTV during beam delivery. As a future perspective, the results may be adaptively utilized for the remaining fractional treatment planning, including the PTV margin adjustment.Written informed consent for the case to be published  was obtained from the patient(s).


Endogenous Lipoid pneumonia is a rare underdiagnosed condition more prevalent in adults. It is usually asymptomatic and a diagnosis is generally made in patients who have become clinically unstable or when an abnormal lung shadow is found on a chest X-ray. In the case here described it was diagnosed by fiberoptic bronchoscopy with bronchoalveolar lavage (BALF) where fat-laden macrophages (oil red O stain) were identified. Since a history of use of oil-based products had been ruled out, amiodarone was deemed to be the most likely cause of lipoid pneumonia. The patient was managed with the replacement of amiodarone with digoxin and treated with oral prednisolone. The patient has remained clinically stable with radiological improvement during a follow-up of two years.We report a case of endogenous lipoid pneumonia secondary to long-term use of amiodarone ( Endogenous lipoid pneumonia is an uncommon clinical condition, more prevalent in adult age, but generally underdiagnosed. It can be associated with bronchial obstruction , systemia decreased diffusion capacity.  for 30 years, systemic hypertension, diabetes mellitus type II and hypothyroidism. Physical examination revealed a well built and nourished patient with pulse rate 69 per minute and blood pressure 110/70 mmHg. Vital signs were within normal range with SpO2 of 95% in room air. Her physical examination revealed bibasal lung crackles, but no evidence of pallor, icterus, cyanosis, clubbing or lymphadenopathy. Her blood work-up was within normal range. Pulmonary function testing demonstrated moderate restrictive lung disease and Since chest X-ray showed bilateral infiltrates, a high resolution computed tomography (HRCT) was conducted. The latter revealed areas of ground glass appearance significantly in the lower lobes of both lungs and airspace consolidations were seen as well  was collected for immunological studies as well. The bronchial washings were sent for smear for acid fast bacilli and cytological examination. No microorganisms were isolated by 48bacteriological examination and no malignant cells were found.The total cell count of the BALF was 287,500/ml. The cells consisted of macrophages (64%), lymphocytes (31%), neutrophils (3%), and eosinophils (2%). The evaluation of BALF with specific staining and coloration showed the presence of fat-laden macrophages (oil red O stain). Figure a decongestant. However, it is described in the literature that chronic use of amiodarone can cause endogenous lipoid pneumonia (phospholipidosis) [The patient had never aspirated or inhaled mineral or vegetable oils and she had never used petroleum jelly intranasally or extra-nasally as pidosis) . AmiodarIn our case, the patient had been taking amiodarone (200 mg OD) for 30 years which was considered the most likely cause. It was therefore discontinued and replaced with digoxin (0.25 mg OD) for the treatment of atrial fibrillation. She was also started on oral prednisolone (20 mg OD) which was gradually tapered over a period of six months.Lipoid pneumonia can be classified into endogenous lipoid and exogenous lipoid pneumonia , 5, 6. CAmiodarone and its metabolites can produce lung damage directly through a cytotoxic effect and indirectly via an immunological reaction. Amiodarone may induce the production of toxic oxygen radicals, which can directly damage cells. In addition, amiodarone appears to promote the accumulation of phospholipids in tissues. There is hyperplasia of type II pneumocytes and a widening of alveolar septae with a cellular inflammatory infiltrate and varying degrees of interstitial fibrosis . On lighThe usual course includes an insidious onset and non-specific respiratory symptoms such as dyspnea and/or cough. Rarely, it may present itself as an acute respiratory illness , 9, 10 eRadiological findings are diverse and may mimic carcinoma, acute or chronic pneumonia, ARDS, or a localized granuloma. High-resolution computed tomography (HRCT) is the best imaging modality for the diagnosis of lipoid pneumonia. However, the radiological manifestations of the disease can be indistinguishable from pneumonia, lung cancer or/ and interstitial lung diseases . In the Treatment of lipoid pneumonia is not well studied and experiences are only limited to case reports. Some authors have suggested the use of systemic corticosteroids in order to slow the inflammatory response.Nevertheless, corticosteroids may not be used routinely and may only be used if the lung injury is severe and ongoing. Some authors have described resection of the most involved lung segments while others the use of immunoglobins or whole lung-lavages , 4, 7–9.In conclusion, endogenous lipoid pneumonia is very uncommon and in this case the diagnosis was attributed to the long-term use of amiodarone.Lipoid pneumonia was confirmed by BALF  where foamy macrophages positive in Oil-red-O staining were found . Withdra


Clinically approved antidepressants modulate the brain's emotional valence circuits, suggesting that the response of these circuits could serve as a biomarker for screening candidate antidepressant drugs. However, it is necessary that these modulations can be reliably detected. Here, we apply a cross-validated predictive model to classify emotional valence and pharmacologic effect across eleven task-based fMRI datasets (n = 306) exploring the effect of antidepressant administration on emotional face processing.We created subject-level contrast of parameter estimates of the emotional faces task and used the Shen whole-brain parcellation scheme to define 268 subject-level features that trained a cross-validated gradient-boosting machine protocol to classify emotional valence  and pharmacologic effect (drug vs placebo administration) within and across studies.We found patterns of brain activity that classify emotional valence with a statistically significant level of accuracy . Our classifier failed to consistently discriminate drug from placebo. Subject population , treatment group (drug or placebo), and drug administration protocol (dose and duration) affected this accuracy with similar populations better predicting one another.We found limited evidence that antidepressants modulated brain response in a consistent manner, however found a consistent signature for emotional valence. Variable functional patterns across studies suggest that predictive modeling can inform biomarker development in mental health and in pharmacotherapy development. Our results suggest that case-controlled designs and more standardized protocols are required for functional imaging to provide robust biomarkers for drug development. •The emotional faces task can evaluate the brain's fMRI response to antidepressants.•We found a consistent signature for emotional valence across 11 such datasets.•We found limited evidence for consistent antidepressant response.•Case-controlled designs and more standardized protocols could increase this yield. The slow onset, unclear biological markers, and variable clinical efficacy even of approved psychiatric drugs makes the potential efficacy of candidate drugs difficult to measure and has led many pharmaceutical companies to withdraw from drug development . BiomarkClinically approved antidepressants modulate the brain's emotional valence circuits, suggesting that the response of these cicruits could serve as a biomarker for screening candidate antidepressant drugs. The emotional faces task has been particularly useful in eliciting the emotional valence circuit . In thisA second advantage of the emotional valence contrast described above is that it can be constructed in either a within or between-subject manner. Duff et al.  have preHere, we apply a machine-learning classifier to a large set of studies of antidepressant effects on brain responses during an emotional faces tasks. We explore the consistency of the emotional valance effect considered both within and between-subjects and the between-subject pharmacologic effect. Because these studies use protocols with considerable variability in scanners, experimental tasks and patient cohorts, we further aim to explore the effect of protocol variability on signature generalizability. To accomplish this, we exploit a dimensionality reduction step  to reduc2For each of eleven datasets, subject-level contrast of parameter estimates of the emotional faces task were created and divided into 268 regions using the Shen whole-brain parcellation scheme. Each region was used as a feature within a cross-validated gradient-boosting machine protocol that classified emotional valence and pharmacologic effect within and across studies. Feature weightings were then mapped onto the brain to allow anatomic localization and visualization.2.1Eleven independent datasets from eight task-based fMRI studies of the effect of antidepressant administration on emotional face processing were available for analysis, representing 306 subjects (See 2.2http://fsl.fmrib.ox.ac.uk/fsl/fslwiki/). The FSL FMRI Expert Analysis Tool (FEAT) was used for general linear modeling (GLM)  package (ng (GLM) . Subject2.3Cognitive models of depression suggest that patients process negative relative to positive stimuli differently from non patients, and that these cognitive processes are causative in the illness. Therefore a contrast looking at the emotional processing circuit activation to negative vs. positive faces may be able to identify illness specific signatures and how the brain's emotional circuits change in response to treatment. We chose a forced-choice gradient boosting machine (GBM) for classification due to its robustness to outliers and its ability to map features back into anatomical brain space .Predictive analyses are prone to overfitting when the number of features far outweighs the number of subjects . Given o2.41)Emotional Valence Classifier. This analysis determined whether and where a signal for emotional valence was consistent enough to discriminate fear from happy face visual conditions. We assessed the performance of the emotional valence classifier with two different types of feature inputs to determine the impact of inter-subject variability and task variability. The first subtracted fear and happy responses within-subject, to account for average differences in visual responses across subjects (i.e. the classifier compared the FvH COPE contrast image to the HvF COPE contrast image). The second compared fear versus fixation COPE files and happy versus fixation COPEs and accounted for across-study differences in task, without being able to minimize individual subject variability in the visual response. Duff et al.  were abl2)drug COPE contrast image to the FvHplacebo contrast image). We further observed whether this signal was consistent across antidepressant dose, frequency, and duration.Pharmacologic Effect Classifier. This analysis used contrasts between Fear and Happy conditions to discriminate patients with drug or placebo protocols within and across studies  are available in Supplementary Methods. In brief: (i) within-study classification: when subjects within one study were considered , the classifier was trained on all but two subjects  and then tested on those held out subjects in an iterative fashion, until the classifier was tested on all subjects; (ii) across-study classification: when subjects across two studies were considered , the classifier was trained on one study and tested on the other. (iii) across-all-subjects classification: when subjects across all studies were considered, to determine our ability to build classifiers that generalize across subjects, the classifier was trained on all but two subjects and then tested on those held out subjects in an iterative fashion, until the classifier was tested on all subjects; (iv) across-all-studies classification: when subjects across all studies were considered , the classifier was trained on all studies except one and tested on that held out study.33.1(i)Within-study classifications (i.e. classifiers trained and tested on different subjects of same study) provide insight into those studies with the most discriminative signals. The five datasets from the Harmer, Murphy and Kumar studies provide the best performance Across-study classifications (i.e. classifiers trained on one study and tested on separate studies) show the ability of classifiers trained on one study to discriminate other studies. We found that results varied considerably depending on the study population evaluated. Classifiers trained and tested on studies of healthy, dysphoric, or major depressive disorder (MDD) performed notably better than studies of low or high neurotic trait  Bonferroni correction for multiple comparisons given 10 classifications per study). We were unable to find a consistent discriminative signal within studies of neurotic subjects. Each train-test dyad may be referenced in (iii)Across-all-subjects classification achieved an average accuracy of 69% (p < .001) in held out data. Classifiers trained and tested on only healthy subjects achieved an average accuracy of 66% .(iv)(iv) Across-all-studies classifications are presented in Supplementary Results.We first describe results associated with discrimination of parameter images subtracting fear and happy responses within-subject, which addresses individual variability in visual responses.In contrast to the above results, we were unable to reliably discriminate the fearful-versus-fixation contrast image from the happy-versus-fixation contrast image better than chance on any classification level .3.2(i)Within-study classification (i.e. classifiers trained and tested on different subjects of same study) performed best on two of the Kumar datasets representing healthy participants  and dysphoric participants . These studies also showed a robust effect for emotional valence.(ii)Across-study classification performed poorly. Overall, clinical state had less of an impact on prediction accuracy than the dose and frequency of antidepressant administration. Studies that used 20 mg of citalopram or escitalopram for 7 days showed a trend towards higher accuracy than studies that administered a single dose of antidepressant.(iii)Across-all-subjects classification achieved an average accuracy of 56% in held out data. Classifiers trained and tested on only healthy subjects achieved an average accuracy of 50%. These poor results may be associated with the fact that drug and placebo sessions were acquired in separate subjects, so could not be subtracted within subjects. (iv) Across-all-studies classifications achieved an average accuracy no better than chance; these results are presented in Supplementary Results.Overall, we found limited ability to identify drug effects in the assessed studies. However, some studies show evidence of a positive drug effect.4We localized an anatomically consistent emotional valence signature in individuals performing the emotional faces task. This valence signature was consistent across subject treatment group (drug or placebo) and drug administration protocol (dose and duration), with similar populations better predicting each other. These results confirm that the emotional valence task strongly probes the brain's valence circuits notwithstanding differences in task design and clinical population. However, we were unable to find a comparably robust signature for pharmacologic effect. An explanation for this can be inferred from the fact that discrimination of emotional valence was poor when the fearful-versus-fixation contrast image was compared with the happy-versus-fixation contrast image, indicating that the effect of emotional valence could not be isolated from, e.g. te effect of face presentation. As these studies assessed drug effects in parallel groups designs, within-subject drug contrasts were not possible. The present results question the extent to which results from parallel group design studies can generalize using a multivariate machine learning approach. Even minor differences across subjects and across drug protocol are likely to alter measurements of antidepressant effects on the brain's functional anatomy assessed using this method.4.1Our significant classification accuracy for emotional valence suggests that happy and fearful faces engage different aspects of the brain's functional anatomy in a spatially consistent way across individuals and studies. In this classification, the most relevant parcels included areas reported in meta-analyses of emotional face processing, namely the amygdala and the fusiform gyrus . While hIn a similar gender-matching emotional faces task, Nord et al.  recently4.2Our classifier failed to consistently discriminate drug from placebo. As a general trend, the classifier performed better when trained and tested on similar drug administration protocols, which used the same dose and frequency. And, overall, drug protocols with higher doses for a longer duration (i.e. 20 mg for 7 days versus 20 mg for 1 day) showed a trend towards higher accuracy. However, we present a guarded interpretation of these results as they could represent false positive results (even though we corrected for multiple comparisons). Why this classification failed could be explained by methodological factors, as well as more general factors that plague drug development studies.When looking for a subtle signal within the brain's large-scale networks, individual variability in brain structure and function understandably becomes a significant confounder. Duff et al.  reportedIn these short, CNS drug administration studies, it is difficult to assess whether therapeutic  CNS drug levels have been reached in each individual due to individual differences in transport proteins that affect blood-brain-barrier permeability . Group-w4.3In the present studies, emotional bias was used to probe the neurobiology of depression based on past group-level observations that depressed individuals have negative emotional bias that corrects with successful treatment . While eThis highlights the fact that there are three ontological levels at play in our study: the clinical constructs of healthy, neurotic, dysphoric, and depressed; the experimental construct of emotional bias; and the etiological construct of receptor-specific treatment targets such as the 5-HT receptor. Each represents a different level of analysis. Using symptom-based clinical constructs to probe etiology-based treatments necessarily muddles group treatment effects; similarly diagnosed patients likely have multiple, diverse etiologies. Neurobiology or etiology-based diagnostic categories would likely help isolate the effect of a mechanism-based pharmaceutical by more logically pairing disease etiology with molecular target .Across these levels, it is unlikely that grouping patients by diagnosis (symptom or etiology-based) is the best way forward because patients may have varying symptoms or etiologies within a diagnostic category. Promising research has shown that within a diagnostic category, patients can be grouped by the presence of a specific cluster of symptoms which then predicts their response to a mechanism-based antidepressant . This suWe suggest that future studies further study the effects of these ontological levels on drug studies and—as much as possible—select more neurobiologically-based means of selecting or probing patient groups.4.4Dimensionality reduction proved a necessary and highly useful step . Whole-bAn unresolved question is which behavioral task and overall study design best captures the normalizing effect of antidepressants in depression. We evaluated the emotional faces task and discovered differences in effect size which could be based on task presentation, subject population, drug administration protocol or a combination of these. While we report progress in this direction, a more concerted study is required to further address these important questions.In summary, we applied a cross-validated predictive model to classify emotional valence and pharmacologic effect across eleven task-based fMRI datasets (n = 306), exploring the effect of antidepressant administration on emotional face processing. We found patterns of brain activity that successfully classified emotional valence, however could not find such patterns for the pharmacologic effect. Our results also suggest that case-controlled designs and more standardized protocols are required for functional imaging to provide robust biomarkers that can help increase the yield of the drug development pipeline.Daniel Barron: No disclosures to report.Mehraveh Salehi: No disclosures to report.Michael Browning: Has received travel expenses from Lundbeck for attending conferences, acted as a consultant for J&J and works part time for P1vital Ltd.Catherine Harmer: CJH has received consultancy fees from P1vital Ltd, J&J, Servier and Lundbeck.Todd Constable: No disclosures to report.Eugene Duff: No disclosures to report.


Natural killer (NK) cells are cytotoxic effector lymphocytes of the innate arm of the immune system with potent activity in eliminating cancer cells. They can directly interact with cancer cells through a specific cell-cell interface termed the immunologic synapse. Activation of NK cell effector function is triggered by an excess of activating over inhibitory signals originating from receptor-ligand interactions between the two conjugated cells. Upon activation, NK cells engage in a series of well-defined cytoskeleton-dependent subcellular events leading to the directed secretion of lytic granules containing effector molecules, such as granzyme B and perforin, toward target cells . In addiin vivo . In turnin vivo . During in vivo , 5. A hain vivo . Preventvia rapid and massive accumulation of filamentous actin at the immunologic synapse, a process which was termed “actin response” (AR; Figure Recently, we reported on the pivotal role of the actin cytoskeleton in driving cancer cell resistance to NK cells . Using ain vivo, and the molecular pathways underlying AR formation and downstream protective effects are under characterization. Our unpublished results support that actin remodeling in tumor cells is a fundamental process at the heart of several immune escape mechanisms, and represents a promising therapeutic point of intervention.Animal experiments have been undertaken to validate the relevance of the AR to tumor immune evasion


Diploid genomes consist of two homologous copies of chromosomes with one from each parent while polyploid genomes contain more than two homologous sets of chromosomes. Most of the reference genome assemblies collapsed haplotypes that represent ‘mosaic’ sequences, ignoring allelic variants that may be involved in important cellular and biological functions. Unzipping haplotypes into distinct sets of sequences has been a growing trend in recent genome studies, as it is an essential tool towards resolving important clinical and biological questions, such as compound heterozygotes, heterosis, and evolution. Herein, we review existing methods for alignment-based and assembly-based haplotype phasing for heterozygous diploid and polyploid genomes, as well as recent advances of experimental approaches for improved genome phasing. We anticipate that full haplotype phasing could become a routine procedure in genome studies in the near future. With the availability of single molecule long-read sequencing, such as PacBio SMRT  sequencing, Oxford Nanopore Technologies (ONT) and Bionano Genomics, as well as high-throughput chromatin conformation capture (Hi-C) technology, most genome sequencing studies can now be completed to chromosomal level assemblies at a fractional cost compared to just several years ago.GRCh38) is derived from 13 volunteers living in New York, and only contains the O allele for the ABO blood group locus. Similarly, for polyploid genomes, a single haploid was often targeted in the reference genome, thereby ignoring a large amount of genetic diversity within the sequenced organism.In most reference genome assemblies, typically two homologous copies of every chromosome, one from each parent, were collapsed together and considered as a ‘mosaic’ reference of the two haplotypes in the current diploid genome assembly approaches. Such reference is called a ‘monoploid’ representation which only reflects a single haplotype throughout the genome. For example, the human reference genome  two decades ago, researchers have been trying to improve the genome assemblies to completely resolve both haplotypes in the same sequencing study. Recent improvements in sequencing technologies and particularly the development of long read sequencing provide opportunities to resolve structural variations between haplotypes that are completely absent in the linear reference genome. In human genome studies, reconstruction of both haplotypes is also clinically relevant, for example, when determining the presence of compound heterozygous mutations, as well as for accurate Human Leukocyte antigen (HLA) typing.cis-regulatory elements, which are able to interact with environmental factors to regulate complex expression networks Another important application of genome phasing is to study allele-specific expression (ASE) or allelic imbalance (AI), which has been suggested as an important mechanism for causing heterosis. ASE or AI reveals a pattern of preferential expression of one parental allele over another Saccharum lineage Additionally, identification of allelic variants can also facilitate research of polyploid evolution and inform crop breeding. The complex polyploid sweet potato genome was first assembled into a consensus reference genome and a novel haplotype phasing method successfully generated a haplotype-resolved genome assembly Haplotype phasing has become a fundamental problem in heterozygous and polyploid genome assemblies. Haplotype phasing is the key for the most accurate representation of genetic composition for a given organism. Herein, we review the existing computational approaches for haplotype phasing – including both alignment-based and assembly-based phasing approaches for heterozygous and polyploid genomes. An overview of the two phasing approaches is illustrated in 2Plummeting cost of Next-generation sequencing (NGS) provided large amounts of data to perform genome-wide scale of haplotype phasing and facilitated the continuous development of phasing algorithms during the past two decades. When the reference sequence is available, the most straightforward method of haplotype phasing is by first aligning the whole genome sequencing (WGS) reads, followed by compiling a set of heterozygous genotypes at polymorphic sites and finally pairing the neighboring haplotypes successively . The exaDifferent alignment-based phasing algorithms have been designed based on different optimization criteria or objective functions, including minimum error correction (MEC), Weighted minimum letter flip (WMLF) and Maximum fragment cut (MFC) A number of haplotype phasing programs based on these algorithms have been reviewed by Je-Keun et al. in 2015 3In contrast to the alignment-based phasing approaches that are mostly targeted at small variants, assembly-based approaches are often more accurate and can cover larger types of genomic variations, such as large indels and structural variations . HoweverEchinochloa crus-galliTo deal with these ambiguities and redundancies, a common practice of assembling heterozygous genomes is to simply reconstruct a single haplotype to represent the whole genome . AssemblAnother automated pipeline, “HaploMerger” was proposed to reconstruct the allelic relationships of contigs in the diploid assembly In addition to the whole genome alignment approaches, read depth-based methods can also be used to determine allelic contigs in heterozygous diploid genome assembly. For a diploid genome assembly with a high level of heterozygosity, a bimodal distribution of read depth is expected which forms the basis to classify the contigs of different origins. The “Purge Haplotigs” program is able to utilize read depth of individual contigs to identify contigs that are suspected to be duplicated sequences, which are assumed to be allelic haplotypes that need to be re-assigned in order to obtain a simple, ‘pseudo-haploid’ reference 4While a single haploid representation is relatively straightforward to derive in heterozygous assemblies, it also throws away a large amount of sequence information that belongs to the other haplotypes. Modern sequencing approaches, such as Single-molecule, real-time Sequencing (SMRT) developed by Pacific Biosciences and Oxford Nanopore Technology (ONT) offers long read length, promising overwhelming performance of assembly for complex genomes de novo genome assembly. Such algorithms seek to go beyond the overly simplistic monoploid representation of the genome, which are especially valuable for heterozygous diploid and polyploid organisms.Currently, Pacific Biosciences technology is often used to assemble many plant genomes, but most of the genome assemblies completed thus far are focused on homozygous individuals or inbred lines, yielding a single representative haplotype. However, for many plant crops that are difficult to be inbred as homozygous individuals, such as many tropical fruit crops, the assembly of heterozygous individuals needs to be handled with additional care. In order to solve this problem, several algorithms are developed to simultaneously reconstruct multiple haplotypes during Arabidopsis thaliana, Vitis vinifera CV (Cabernet Sauvignon) and highly heterozygous wild diploid Clavicorona pyxidateThe FALCON and FALCON-Unzip algorithm provide a clean solution that enables sequential assembly of the original sequencing data and ongoing identification of the phased diploid genome Bos taurus taurus and Bos taurus indicus were sequenced and fully resolved into two parent haplotypes, with a NG50 of haplotig size of over 20 Mb with 99.998% accuracy, which exceeds the quality of current bovine reference genome a priori as well as sequenced in order the phase the F1 genome. This requirement not only results in higher cost, but also can be limiting in scenarios such as when the plant species was either collected from the wild or has less developed breeding programs so that the parentage information may not be available.A more recent approach, “trio binning” is proposed to simplify haplotype assembly by addressing allelic variation prior to assembly 5in situ to obtain interacting DNA fragments. The ligated DNA fragments are captured by biotin and then sequenced by paired-end sequencing. The interacting DNA fragments, shown as pairs of linked reads, reveal long-distance information about the grouping and linear organization of sequences across the entire chromosomes Genome assembly incorporating PacBio long reads sequencing and proximity ligation-based methods is an efficient approach to construct chromosomal level genome assembly. High-throughput chromatin conformation capture (Hi-C) is a technology derived from chromatin conformation capture (3C) technology, which combines chromatin proximity ligation method and high-throughput sequencing to obtain a fine map of chromatin interaction across the whole chromosome de novo assemblies of the genome projects in mammals, plants and insects. Hi-C data can also be used to phase genome onto separate haplotypes at chromosome-scale since homologous chromosomes occupy distinct territories in nuclei In the past decade, Hi-C based assembly approaches have become broadly available to generate reliable chromosome-scale One recently published genome phasing and scaffolding software is FALCON-Phase developed by Phase Genomics However, FALCON-Phase pipeline is designed for phasing diploid genomes and does not yet support the construction of chromosomes for polyploid genomes. To address this limitation, a new pipeline of Hi-C scaffolding named ALLHiC was specifically developed for polyploid genomes 6The initial Human Genome Project was primarily carried out through the hierarchical sequencing of large-insert clones with long DNA fragments (50–200 kb) inserted in bacterial artificial chromosomes (BACs) Linked-Reads, a new sequencing technology developed by 10x Genomics, holds the promise to whole genome haplotype phasing at a relatively low cost. This technology leverages microfluidics to partition and barcode high molecular weight DNA molecules, which are then subject to Illumina short-reads sequencing. Paired reads that contain the same barcode are considered to be derived from the same haplotype and linked together during the genome assembly process, which greatly reduces the complexity of genome assembly. Using the Linked-Reads technology, a recent research study on human genomes reveals a comprehensive list of alternative haplotypes in diverse populations de novo assembly Another related technology, single-tube long fragment read (stLFR) technology developed by Beijing Genomic Institute (BGI), recently showed its potential in haplotyping and Clones, linked reads and long fragment reads all provide long-range linkage evidence in the range of hundreds of kilobases to several megabases in length in order to support the phasing of haplotypes – extending well beyond the range that are typically offered by a single WGS read including the latest nanopore reads. Both alignment-based and assembly-based approaches are capable of utilizing these experimental evidence during genome phasing. The popularity of the experimental approaches for phasing would be largely determined by their respective throughput and cost.7S-locus that are known to be structurally diverse. The second class of haplotype phasing is assembly-based haplotype construction. For this strategy, full-length sequences for individual haplotypes are produced, which are often the preferred phasing method when available due to the higher resolution provided than the alignment-based phasing.Even though there are a number of existing approaches, both computational as well as experimental, it is still challenging to achieve complete and fully accurate haplotype phasing. Currently, there are two overall strategies for haplotype phasing that we reviewed . The firCurrently, most state-of-the-art algorithms employ the alignment-based strategy to determine adjacent loci based on Illumina short reads and/or PacBio long reads For the assembly-based strategy, newer generation of genome assemblers such as FALCON-Phase and ALLHiC, are able to separate allelic contigs and produce chromosomal scale of genome assemblies for heterozygous diploid or polyploid genomes De novo assembly of a human genome using HiFi reads produced a contiguous and accurate genome with a contig N50 of over 15 Mb and consensus accuracy of 99.997% Further developments on haplotype phasing and haplotype-resolved genome assembly techniques are needed The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


A recent cohort study among Papua New Guinean women surprisingly showed iron deficiency during pregnancy to be associated with increased birth weight. These findings seemingly contradict previous trial evidence that iron supplementation leads to increased birth weight, particularly in iron-deficient women, and hence require explanation.We have re-analysed data from a previous trial in Kenya and demonstrated that, because women who were initially iron deficient respond better to iron supplementation, they show an increase in birthweight. There is evidence that this benefit is decreased in iron-replete women, possibly due to the adverse effects of haemoconcentration that can impair oxygen and nutrient transfer across the placenta. The Papua New Guinean results might be explained by a similar differential response to the iron supplements that they all received.Antenatal iron supplementation should ideally be administered in conjunction with measures to prevent, diagnose and treat malaria given the propensity of pathogenic microorganisms to proliferate in iron-supplemented individuals. However, even where services to prevent and treat malaria are poor, current evidence supports the conclusion that the benefits of universal iron supplementation outweigh its risks.https://bmcmedicine.biomedcentral.com/articles/10.1186/s12916-018-1146-z.Please see related article: https://bmcmedicine.biomedcentral.com/articles/10.1186/s12916-019-1376-8.Please see related article: The online version of this article (10.1186/s12916-019-1375-9) contains supplementary material, which is available to authorized users. A recent cohort study in Papua New Guinean pregnant women reported an association between iron deficiency (ferritin concentration < 15 μg/L) and higher birth weight by 230 g), particularly among primigravidae [30 g, parThe WHO recommendation was based on a meta-analysis of randomised trials in which antenatal iron supplementation was suggested to lead to a slight increase in birth weight  [Plasmodium infection. These markers were used to calculate the body iron index, i.e. the natural logarithm of the ratio of the adjusted ferritin concentration to the adjusted transferrin receptor concentration. This quantitative indicator has been shown to be linearly associated with the size of the body iron store in iron-replete individuals, and with the size of the functional deficit that would need to be corrected before iron could again be accumulated in the store in an iron-deficient individual. Multiple fractional polynomial regression analysis showed that the birth weight response to administered iron decreased with initial iron status . All women in the study were prescribed daily supplementation with iron and folic acid. Consistent with our findings in Kenyan women, iron-supplemented women who were initially iron deficient had newborns with a higher birth weight than their peers who were initially iron replete. What could explain this phenomenon?In normal pregnancies, plasma volume expansion leads to a gradual reduction in haemoglobin concentration and haematocrit levels in the gestational age period between 7 and 36 weeks, despite concurrent increases in the total erythrocyte and haemoglobin mass . This haIn iron-deficient women, haemodilution without an adequate supply of iron to the developing erythrocyte and haemoglobin mass will likely result in placental insufficiency, leading to low birth weight and prematurity. This may explain the negative association that we found between iron status and birthweight in women who were not supplemented with iron  without an apparent effect on P. falciparum infection risk [There is ample evidence that iron supplementation predisposes towards the proliferation of pathogenic microorganisms. In vitro studies indicate that asexual blood stages of ividuals , 9. Thesothelium , thus avion risk .Randomised trials offer superior methodology and should be given higher priority in the hierarchy of evidence than cohort studies because they minimise the risk of selection bias. Benefits of universal iron supplementation in pregnancy are likely to vary with the prevalence of iron deficiency  and are Plasmodium infection may also improve the absorption of non-haem iron from supplements or dietary sources [Antenatal malaria prevention not only results in direct health benefits to mothers and neonates, but has the additional advantage that elimination of inflammation associated with chronic  sources .The efficacy and coverage of preventive measures against malaria in pregnancy are generally low. For example, among African countries that reported on intermittent preventive treatment in pregnancy in 2016, only 19% received the recommended minimum of three doses of intermittent preventive treatment . It seemAdditional file 1:Methods to assess the influence of maternal body iron status at baseline (i.e. second trimester of pregnancy) on the magnitude of the effect of antenatal iron supplementation on neonatal birth weight. (DOCX 19 kb)


Atherosclerosis is a chronic immunomodulated disease that affects multiple vascular beds and results in a significant worldwide disease burden. Conventional imaging modalities focus on the morphological features of atherosclerotic disease such as the degree of stenosis caused by a lesion. Modern CT, MR and positron emission tomography scanners have seen significant improvements in the rapidity of image acquisition and spatial resolution. This has increased the scope for the clinical application of these modalities. Multimodality imaging can improve cardiovascular risk prediction by informing on the constituency and metabolic processes within the vessel wall. Specific disease processes can be targeted using novel biological tracers and “smart” contrast agents. These approaches have the potential to inform clinicians of the metabolic state of atherosclerotic plaque. This review will provide an overview of current imaging techniques for the imaging of atherosclerosis and how various modalities can provide information that enhances the depiction of basic morphology. In the later stages, atherosclerosis may manifest as either exertional symptoms due to luminal narrowing or sudden events related to plaque rupture. The latter may cause local occlusion, as occurs in myocardial infarction. Alternatively, sudden rupture of an atherosclerotic plaque may cause distal embolism. This is typically seen in ischaemic stroke distal to a proximal internal carotid artery plaque rupture. Atherosclerosis is a chronic immune-modulated pathological process affecting multiple vascular beds and leads to diseases that are a major cause of morbidity and mortality worldwide.2 Combining established imaging strategies with “smart” contrast agents and biological tracers allows us to obtain complementary assessments of biological activity within plaques. This carries the potential to improve our pathophysiological understanding, leading to better patient risk stratification and management.Structural vascular imaging throughout the body has advanced rapidly. Certain morphological features of atherosclerotic plaques are associated with a higher risk of disease progression.This review will focus on the techniques available to image atherosclerotic disease. We will describe how certain modalities are well suited to image specific cardiovascular components. Finally, we will illustrate how studying biological activity within plaque advances our ability to detect disease, assess risk and guide patient management.3 Exposure to established risk factors, such as hypertension, hypercholesterolaemia and smoking, creates a systemic environment that encourages endothelial dysfunction,4 oxidation of lipoproteins,5 production of free oxygen radicals6 and leukocyte migration.7 Accumulation of oxidized lipoproteins within the vessel wall forms fatty streaks. Macrophages migrate across the endothelium to phagocytose these lipid-rich proteins. The high intracellular cholesterol content of macrophages induces pathways of cell death. The resultant debris, along with necrotic endothelial and smooth muscle cells, forms the principal constituent of the lipid-rich core  macrophage-rich fibrous cap and microcalcification.11 The initiation of vascular calcification occurs on a microscopic level  and involves the deposition of calcium and phosphate-rich hydroxyapatite crystals.12 This early stage of “microcalcification” signifies intense biological activity and is associated with increased plaque vulnerability. In contrast, the larger established deposits of macroscopic calcification that ultimately develop are associated with plaque stability and a more quiescent phase of disease.Calcium deposition in vessel wall inflammation occurs as a macrophage-mediated reparative response to oxidized lipid deposition and endothelial dysfunction. However, rather than being a passive by-product of degradation, the process of vascular calcification is active and controlled. Calcification in atherosclerosis primarily affects the intima. This is in contrast to medial calcification typically observed in patients with diabetes or chronic kidney disease, which adopts a concentric transmural morphology.13 Rupture of the thin fibrous cap exposes the lipid-rich necrotic core to luminal flowing blood, initiating rapid and aggressive thrombosis that can cause vascular occlusion. However, plaque rupture is frequently silent and subclinical. Over time, vascular remodelling incorporates the exposed thrombus and the degree of stenosis may worsen.Vulnerable plaques have a thin fibrous cap that is devoid of vascular smooth muscle cells and exhibits intense macrophage recruitment.14Angiogenesis and collateralization can partially compensate for reduced blood flow caused by increasingly stenotic vessels. However, if arterial disruption is rapid, this compensatory angiogenesis has had insufficient time to occur, and the resultant ischaemia is more severe. Thus, vulnerable plaque rupture may cause sudden arterial occlusion, loss of tissue perfusion and catastrophic end-organ infarction. Prompt therapeutic reperfusion is required to minimize irreversible tissue loss in a highly time-dependent process.Anatomical imaging is diverse and flexible. The chief objectives are to detect luminal narrowing and characterize atherosclerotic plaque. Imaging also allows a global assessment of atherosclerotic disease burden within the entire vascular territory. This information helps clinicians stratify the risk of future adverse events.Catheter-based contrast angiography is the most frequently used method to image the coronary vessels. This is largely because of the high spatial and temporal resolution that can be obtained. Indeed, catheter angiography is also a platform for intervention. However, catheter angiography alone merely provides a “lumenogram” of the coronary circulation. Plaque characterization is possible but requires specialist equipment. Intravascular optical and ultrasound imaging allow the constituents of plaque morphology to be assessed in remarkable detail. These techniques are an excellent choice to image high-risk patients who may require simultaneous intervention.There is a need to identify adverse plaque features using non-invasive imaging techniques. This is especially true in low- and medium-risk patient cohorts. Modern cardiovascular medicine has seen increased adoption of non-invasive imaging techniques. Angiography by CT or MRI is safe and offers a reliable alternative to catheter angiography. These imaging modalities can simultaneously image the vessel wall and any surrounding atherosclerosis. Similarly, ultrasound can determine blood flow across a lesion to quantify stenosis. Spectral analysis and specialized contrast agents can characterize plaque at the same time. Non-invasive imaging may also improve clinical decision making. It separates diagnosis from intervention, thus allowing rational decision making by a multidisciplinary team.Here we discuss the imaging techniques available to visualize morphological features of atherosclerotic vessels. We touch briefly on how these techniques can help characterize plaque.Intravascular imaging techniques have the benefit of capturing images from close proximity to the plaque, thus offering extremely high-resolution images.15 High signals in the fibrous cap signify macrophage adherence and thrombus formation.16 These high resolution benefits need to be balanced against reduced tissue penetration which limits assessment of deeper plaque constituents. OCT is used clinically to detect vulnerable plaques and guide therapy in the coronary arteries.17,18 OCT is now being used for these purposes in carotid artery stenosis19 and peripheral vascular disease.20Optical coherence tomography (OCT) provides exceptionally detailed images of the fibrous cap using near infrared light delivered via a fibre optic wire . The OCT21Near infrared spectroscopy (NIRS) is another catheter-based invasive technique. NIRS does not require a blood free field and uses wave scatter to produce a gradient map corresponding to the probability of adjacent lipid . The res22,23 Despite reasonable tissue penetration, IVUS lacks the spatial resolution to measure the thickness of the fibrous cap. Like conventional ultrasound, mineralized calcium deposits cast acoustic shadows, thereby obscuring underlying tissue detail.IVUS is a more established and, consequently, cost-effective technique than OCT and NIRS. It packages a high-frequency ultrasound probe in a catheter that can directly visualize adjacent atherosclerotic plaque from within the lumen . High-re15,24 Its use has been validated to perform a similar function in the carotid25 and lower limb26 arteries.NIRS/IVUS is well established in detecting and treating coronary disease.27 and the greyscale pixel intensity of atherosclerotic plaque images have been shown to correspond with histological features.28 Ultrasound is non-invasive, radiation-free and portable. Hence, it is often used as the first-line imaging modality to quantify stenoses throughout the peripheral vascular tree.In superficial vessels, such as the carotid and limb arteries, duplex ultrasound combines structural and functional data to quantify the degree of stenosis caused by atherosclerotic plaque. The ratio of peak systolic velocity proximal and distal to suspected lesions is used to estimate the degree of stenosis. Ultrasound can be used to measure the total plaque area29 Detecting this thickening using ultrasound can be used as a "window" to an individual’s global cardiovascular health. Indeed, increased IMT is associated with significantly increased risk of myocardial infarction, stroke and death.30The high resolution of ultrasound can differentiate components of the arterial wall in superficial vessels. Pathological proliferation of the intimal-medial layer is a sign of early subclinical plaque formation.e.g. SF6) administered in the venous space can highlight specific features of arterial atherosclerotic plaque. In the carotid arteries, microbubbles identify neovascularization in culprit lesions with a sensitivity and specificity greater than 80%.31 In patients with abdominal aortic aneurysms, contrast-enhanced ultrasound provides real-time characterization of luminal flow. Visualization of complications following endovascular repair, such as endoleaks, is feasible.19,32 The lack of ionizing radiation makes contrast-enhanced ultrasound an ideal modality for situations that require repetitive imaging.Contrast agents containing a homogenous suspension of inert gas microbubbles  is well established to assess the cardiovascular system. It is non-invasive and accessible. CTA has the added benefit of visualizing the entire vessel from its origin to target structure, even in tortuous vessels. Among its many applications, CTA has the spatial resolution to detect focal luminal stenoses and provide a global assessment of vascular disease. Owing to the short acquisition times of new generation CT scanners, it is now possible to image the coronary vessels in great detail. Indeed, CTA is highly accurate in determining coronary stenosis severity and has been incorporated within many clinical guidelines as the first-line imaging modality for individuals with suspected cardiac chest pain. CTA is used throughout the vascular tree. In the carotid arteries, CTA has a reported sensitivity approaching 100% and specificity of 63%  to detect a stenosis greater than 70%.35–37 The napkin-ring sign on centreline reconstruction of the coronary vessels represents differentiation between the fibrous plaque and necrotic core.38 It is characterized by a crescentic high attenuation pattern surrounding a low-attenuation atheromatous lesion distinct from the vessel lumen—a morphological pattern that mirrors plaque histology.Beyond the assessment of luminal stenosis, CT can characterize plaque morphology. Several high-risk plaque features such as positive remodelling, spotty calcification, high-attenuation fibrous plaques and low-attenuation lipid-rich necrotic plaques can be identified.—particularly in small calibre vessels with a high burden of calcification. In acute disease, CT is unable to differentiate soft-tissue components of a plaque. For instance, it is not possible to distinguish directly a stable fibroatheromatous lesion from acute plaque haemorrhage or thrombosis. Identification of the culprit plaque relies on a combination of radiological features along with the clinical presentation and pattern of end-organ damage.CT is particularly well suited to visualize vessel calcification. However, beam hardening in relation to densely calcified plaque causes blooming that exaggerates plaque size and obscure the lumenT1 weighted, T2 weighted, proton density) offers excellent soft tissue characterization. This allows the constituents of atherosclerotic plaque to be investigated without the need for ionizing radiation. These properties make MRI useful in the longitudinal studies of chronic cardiovascular diseases. Administration of gadolinium (Gd)-based contrast media improves image acquisition times and provides further structural information outlining differences between the blood pool and vessel wall.39Angiography using MRI is best suited to imaging large stable vessels such as the carotid arteries. Multicontrast MRI  are 30 nm iron oxide nanoparticles stabilized with low-molecular-weight dextran. USPIOs accumulate in macrophages following phagocytosis and remain in the circulation for extended periods.neurysms .50 Using1H), 31-Phosphorus (31P), and 13-Carbon (13C).51In vivo carotid studies using MRS have successfully quantified cholesteryl esters within atherosclerotic plaque.46,52 Cholesteryl esters are the major class of lipids found in the lipid-rich necrotic core of vulnerable plaques. The chemical composition of structures is obtained using a chemical shift imaging sequence to acquire spectra over and around the atherosclerotic plaque. MRS amplitudes for specific metabolites, such as lipids, are then interpreted as a ratio to the amplitude of intrinsic water.53,54 The final analysis allows detection and quantification of the lipid content of atherosclerotic plaque.MR spectroscopy (MRS) combines the spatial imaging obtained from MRI with spectral analysis to detect the chemical composition and metabolic state of cardiovascular tissue. MRS is able to detect a range of atoms, including 1-Hydrogen  and single proton emission computed tomography (SPECT) to detect increased activity of specific disease processes, such as increased glycolytic activity or microcalcification.Biologically active atherosclerotic lesions are inherently unstable and prone to rupture.Biological radiotracer molecules typically consist of two components. One section has a ligand that targets sites of specific disease activity. The other component consists of a radioisotope. PET and SPECT scanners can detect the intensity and distribution of radiotracer activity following molecular engagement with target disease processes. PET has superior image definition and has gained popularity over SPECT.56Molecular imaging in cardiovascular medicine has made substantial advances recently with an ever-expanding array of biological tracers targeting different processes. Approaches to standardize quantification of radiotracer uptake has improved the reporting and reproducibility of results.18F-Fluorodeoxyglucose (18F-FDG) is a glucose analogue. It is the most commonly used biological tracer in clinical practice. 18F-FDG is taken up by metabolically active cells and its immediate metabolite is trapped within the cell following phosphorylation. This allows the quantification of cellular glycolytic activity. In vascular inflammation, uptake of 18F-FDG is pronounced in smooth muscle cells, endothelial cells and macrophages.57 Increased 18F-FDG uptake in carotid and coronary atherosclerosis correlates with unstable plaques and those that exhibit histological features of vulnerability,58,59 including increased CD68 macrophage density.6018F-FDG reflects glycolytic activity, uptake is non-specific. This limits interpretation of 18F-FDG PET in structures adjacent to those with a high physiological affinity to glucose, such as the myocardium. In order to visualize 18F-FDG PET uptake in the coronary arteries, it is necessary to suppress myocardial uptake using a low-carbohydrate, high-fat preparatory diet. However, these attempts at myocardial suppression are ineffective in around a quarter of patients, rendering assessment of coronary plaque activity very challenging.61Since 18F-FDG. Here, the combination of a somatostatin ligand with a DOTA-  or NOTA-  based “cage” has resulted in the development of various tracers. The cage houses a positron-emitting isotope, such as Gallium-68 (68Ga) or Copper-64 (64Cu). PET studies reveal that these agents exhibit preferential vascular uptake in patients with established cardiovascular risk factors and adverse Framingham risk scores.62Tracers that target macrophages directly, such as the somatostatin subtype 2 (SST2) receptor analogues, overcome the limitations caused by the poor specificity of 63 Ga-DOTA-TATE  uptake reveals selective binding of the radiotracer to CD68-positive macrophage-rich lesions. In a pilot study,63Ga-DOTA-TATE correctly identified metabolically active coronary and carotid lesions with good reproducibility and higher sensitivity than 18F-FDG.64Histological comparison of carotid plaque with high11C-choline and 18F-fluthymidine. Chief amongst these are translocator protein (TSPO) ligands (11C-PK11195), which are 18 kDa proteins expressed in the mitochondria of most cells. Activated macrophages exhibit marked upregulation of these translocator proteins. Preliminary studies suggest that 11C-PK11195 PET/CT correctly identifies suspected carotid lesions and has favourable tissue-to-background ratios compared to 18F-FDG.65Other tracers targeting macrophage activity include vascular cell adhesion molecule-1 (VCAM-1), 63Ga-Pentaxifor, can be used to target these receptors.66 In the coronary arteries,63Ga-Pentaxifor is high in culprit vessels following acute myocardial infarction . These lesions also have high concentrations of CD68 +macrophages.67Activated inflammatory cells within atherosclerotic plaque exhibit CXCR-4 receptors. Novel PET radiotracers, such as18F-Sodium fluoride (18F-NaF) binds to exposed hydroxyapatite crystals. Due to surface area effects, it preferentially binds to areas of developing microcalcification, which is beyond the resolution of CT.63,68 Because 18F-NaF is not taken up by myocardium, the background signal is low, which allows even relatively low signal to be visualized within the coronary vessels  is a biological tracer that concentrates in hypoxic viable cells due to an accumulation of its metabolites in an oxygen-deprived environment. Animal models show 18F-MISO correlates well with aortic atherosclerosis and areas of FDG uptake.71 Exploratory human studies suggest that 18F-MISO PET/CT corresponds to areas of vessel hypoxia, increased macrophage density and 18F-FDG uptake in symptomatic carotid artery disease.68,72vβ3 integrin cell surface glycoproteins. In animal models, αvβ3 targeted imaging with MRI sensitive paramagnetic particles identify areas of angiogenic proliferation.73 A novel PET tracer, 18F-Galacto-RGD, can also target αvβ3 integrin74 and in mice models of atherosclerosis, binds to sites of new atherosclerotic plaque. However, histological analysis in these mice models showed that uptake co-localized in macrophage-rich atherosclerotic plaque, as opposed to angiogenesis specifically.75 Culprit carotid plaques demonstrate increased 18F-Galacto-RGD uptake on pre-operative PET/CT and post-operative autoradiography analysis. In a small sample of patients, 18F-Galacto-RGD binding showed a tendency to bind to plaque rich in macrophages and those with increased vasa vasorum density.76Angiogenic endothelial cells and hypoxic macrophages in atherosclerotic plaque express αin vivo detection of high-risk features and culprit plaques  and now recommends CTCA as the first-line investigation in patients with stable chest pain of suspected cardiac origin. The SCOT-HEART trial showed that, in patients with stable chest pain, CTCA reclassifies the diagnosis of coronary artery disease in up to a quarter of patients and improves the accuracy of diagnosing angina.79 The coronary artery calcium (CAC) score is a non-contrast CT technique that estimates overall calcification in vascular beds. The CAC score provides an improvement in predicting future cardiovascular events in patients at medium risk of ischaemic heart disease (Framingham risk score >10%).80 In particular, CAC scores capture patients at high risk, who would otherwise be incorrectly classified using traditional scoring methods.81Coronary stenosis alone does not predict the risk of future cardiovascular events. Instead, events appear most closely related to adverse plaque features and total burden: the more plaque you have, the more likely one will rupture and cause a myocardial ischaemia.p = 0.001).82 In the heart, PET has traditionally remained a challenging area due to the non-specific uptake of 18F-FDG by the surrounding myocardium.69 However, uptake of 18F-NaF is more selective to regions of microcalcification. This makes it an ideal agent for studying the coronary arteries. In a study of 80 patients, 18F-NaF preferentially bound to IVUS-confirmed culprit coronary plaques.69 Similarly, a study of 119 participants showed that 18F-NaF PET/CT findings correlated with underlying angina (p = 0.023), prior cardiovascular events (p = 0.016) and Framingham risk scores (p = 0.011).83 The predictive power of 18F-NaF to identify culprit coronary plaques is the focus of an ongoing prospective multicentre trial in patients with recent acute coronary syndrome and multivessel disease: the PRE18FFIR study (NCT02278211). Whether this can be enhanced by other novel radiotracers, such as 68Ga-DOTATATE,84 remains to be established.Multimodality imaging adds a further dimension to the assessment of patients with chest pain. For instance, in patients with acute coronary syndrome, the lipid core burden index on NIRS independently predicts future cardiovascular events  inhibitors. However, the added value of such imaging will need to be established in prospective trials, such as the DIAMOND study (NCT02110303) where the benefit of dual antiplatelet therapy in patients with high coronary 18F-NaF PET uptake is being assessed.Non-invasive multimodality imaging enables the detection of high risk features 85 and ECST86 studies showed the benefit of endarterectomy in symptomatic high-grade carotid artery stenosis.87 Ultrasound is the mainstay of risk stratification of symptomatic carotid artery disease.88Extracranial carotid plaque rupture results in acute thrombus formation that may occlude the internal carotid artery or be the lead point for dissection. The thrombus may embolize to the brain or the eye causing permanent tissue loss. The goal in treating carotid artery disease is to prevent future thromboembolic events. Imaging the carotid arteries fulfils an important role in cerebrovascular risk prediction. The NASCET89 However, nearly a third of recurrent disabling or fatal strokes occur in patients with internal carotid artery stenosis less than 50%.86 Ultrasound, CT and MR are limited to studying the morphological features of atherosclerotic plaque. They cannot reliably predict future cerebrovascular events in “low” risk groups with lesser degrees of stenosis.89We now know that early surgical intervention following an index carotid artery event is important to reduce recurrent embolic episodes from carotid artery lesions. Patients may have tandem disease. This group was excluded from the original NASCET trials. Hence, many guidelines do not advocate duplex ultrasound as the sole imaging arbiter prior to surgery. CT and MR angiography have the benefit of overcoming vessel tortuosity and visualizing the entire artery.18F-FDG or 18F-NaF PET can detect the biological activity of culprit plaques, with 18F-NaF in particular binding to culprit carotid lesions characterized by a lipid-rich necrotic core  will assess the clinical significance of 18F-NaF PET/MRI in relation to plaque vulnerability, the requirement for surgery and clinical outcomes. This may lead to better risk stratification of patients with stroke permitting better selection for surgery: avoiding unnecessary carotid endarterectectomy whilst encouraging intervention in high-risk plaque below the current threshold for surgical intervention.The treatment of asymptomatic patients with carotid artery disease remains controversial.91 although many advocate a lower threshold for females. Endovascular or open surgical repair is considered once aneurysms reach this threshold.92The pathobiology of abdominal aortic aneurysms shares key features with atherosclerotic disease. Ultrasound scan remains the cornerstone of abdominal aortic aneurysm screening and surveillance. However, current prediction models rely on a single morphological measurement of anteroposterior diameter to prognosticate the risk of aneurysm rupture. In males, this diameter is 55 mmSome aneurysms may rupture at sizes smaller than 55 mm whilst others reach large dimensions without causing any harm. Like carotid artery disease, multimodality imaging may provide better risk stratification than ultrasound scan-derived diameter alone.T1-signal is seen in disorganized thrombus that is typically unstable.93 A series of 35 patients with abdominal aortic aneurysms found that individuals with high thrombus T1 signal intensity were likely to exhibit twice as fast aneurysm growth compared to those with organized thrombus.94 It is uncertain whether thrombus formation accelerates aneurysm growth. One possible explanation is that organized thrombus is the product of chronic stable aneurysm morphology, whereas a rapidly growing aneurysm has a large turnover of thrombus that is inherently unstable.MRI signal intensity can characterize thrombus organization with high accuracy. A high 48 In aneurysmal disease, 18F-NaF PET/CT detects aortic microcalcification. This is a common end point of multiple pathological processes ultimately leading to aortic degeneration. Increased 18F-NaF uptake is associated with faster aneurysm growth and an increased risk of rupture or requiring repair, independent of aneurysm diameter.70Smart contrast agents such as USPIO can further characterize biological activity within the aneurysm wall. High aortic mural USPIO uptake localizes to areas of macrophage infiltration and reflects vessel wall inflammation. An increased burden of USPIO uptake is observed in aneurysms with faster rates of expansion.Timely intervention is invaluable prior to the catastrophic consequences of aneurysm rupture. Conversely, better risk prediction may aid the decision of conservative aneurysm management in patients with major comorbidities. Much like carotid disease, multimodal imaging may provide useful insights into future risk and be a better guide for surgical intervention in patients with aneurysms above and below the current standard of care to consider intervention at a binary cut point of aneurysm diameter above 55 mm.Peripheral vascular disease is a heterogenous group of occlusive arterial conditions involving the lower limbs. Atherosclerosis primarily causes intimal calcification. In contrast, diabetes mellitus and chronic kidney disease cause arterial stiffness by enhancing transmural calcification of the medial layer of the artery. Both processes are initiated by microcalcification.Arterial duplex, MR and CT angiogram can visualize the lumen in large vessels but may either over estimate or underestimate the degree of stenosis in narrower arteries, such as below the knee. For instance, calcification in these small vessels causes a blooming artefact in CT Angiograms that obscures the view of the lumen. When calcification is not present, a contrast-enhanced residual lumen can be over estimated in CT with voxels that are too large. Hence, diagnostic angiography remains the gold-standard to visualize luminal narrowing in the lower limbs owing to its high spatial resolution. This approach, however, does not image the arterial wall or inform on the biological activity within it.18F-Fluoride binds with good affinity to femoral plaque and is better at detecting inflammation compared to 18F-FDG.95 A study of 409 oncology patients undergoing 18F-NaF PET/CT were incidentally found to have significant femoral artery 18F-fluoride uptake. The degree of uptake correlated strongly with hypertension, hypercholesterolaemia, smoking habit, diabetes mellitus and prior cardiovascular events.96 The clinical progression of this uptake in longitudinal studies remains to be validated.Multimodal imaging can provide objective surrogate end points to confirm target engagement and treatment efficacy in relatively modest-sized populations.97–99 and non-invasive100–102 imaging techniques. Intravascular imaging using OCT and IVUS was used to study the effects of statins on atheroma in trials such as ASTEROID,97 ESTABLISH99 and REVERSAL.103 Similarly, MRI has been used to quantify the reduction in the lipid content of necrotic core after treatment with rosuvastatin at 1 and 2 years of follow-up.100,101 The ATHEROMA trial used USPIO-enhanced MRI to show a reduction in carotid plaque inflammation within 3 months of starting atorvastatin.102One example is the study of statins in cardiovascular disease. The effect of statins on atherosclerotic burden has been extensively investigated using invasive104 Conversely, MRI and PET/CT have been used to determine a lack of clinical efficacy. The dal-PLAQUE study did not show a reduction in vessel wall inflammation using the CTEP inhibitor, dalcetrapib: an agent that is now known to be clinically ineffective.105Imaging may be used to validate the effect of therapeutic interventions. The REMNANT trial studied the effect of thrombus aspiration from culprit plaques in patients with acute coronary syndrome. The investigators used IVUS to quantify the percentage increase in lumen volume following thrombus aspiration and showed that aggressive intervention was likely to result in successful stent deployment.Detailed structural and functional assessment of the vascular tree is possible with conventional imaging modalities. However, the promise of identifying vulnerable atherosclerotic plaque leading to cardiovascular events remains elusive. Multimodality cardiovascular imaging provides the clinician with valuable information on the morphology and metabolic state of atherosclerotic diseases. Combining imaging modalities offers powerful tools to study specific disease processes in the context of diagnosis, risk prediction and targeted intervention.A paradigm shift to move away from the assessment of stenosis severity alone is required. Detecting high-risk plaque features promises to reclassify low- and medium-risk individuals who are, in fact, at high risk of developing adverse cardiovascular events. Correctly identifying this group is the greatest unmet clinical need in contemporary cardiovascular imaging. Incorporating novel imaging techniques addresses the limitations of modern stenosis-driven approaches. Translating these wide range of imaging techniques into clinical practice is the next major advance in atherosclerotic plaque assessment. Ongoing research on the role of metabolic imaging promises to detect and prognosticate cardiovascular disease progression. These studies will better inform the translation of novel imaging modalities to mainstream clinical practice.


It has been almost a decade since the 2009 influenza A virus pandemic hit the globe causing significant morbidity and mortality. Nonetheless, annual influenza vaccination, which elicits antibodies mainly against the head region of influenza hemagglutinin (HA), remains as the mainstay to combat and reduce symptoms of influenza infection. Influenza HA is highly antigenically variable, thus limiting vaccine efficacy. In addition, the variable HA head occupies the upper strata of the immunodominance hierarchy, thereby clouding the antibody response toward subdominant epitopes, which are usually conserved across different influenza strains. Isolation of monoclonal antibodies from individuals recognizing such epitopes has facilitated the development of recombinant vaccines that focus the adaptive immune response toward conserved, protective targets. Here, we review some significant leaps in recombinant vaccine development, which could possibly help to overcome B cell and antibody immunodominance and provide heterosubtypic immunity to influenza A virus. Influenza viruses belong to the family of Orthomyxoviridae and consists of A, B, C, and D types. Types A and B are currently circulating among humans –4. InfluHA is the most abundant glycoprotein on the influenza virion surface and is crucial for host viral entry by binding to the terminal sialic acid residues on epithelial cells, resulting in fusion of viral and host cell membranes. HA is a trimer consisting of a globular head, harboring the receptor binding site, and an elongated stem region . Even thHA stem has been an important candidate for development of universal vaccines because the stalk region is relatively conserved and evolves much slower and accommodate less amino acid substitutions as compared to the head domain. This could be due to minimal antibody pressure from low amount of circulating anti-stem antibodies , 24 and HA stem antibodies protect by (i) preventing viral entry by blocking the fusion of host cell membrane and viral membrane , (ii) reIn humans, memory B cells (Bmem), and antibodies against HA stem are subdominant and present in low levels. Analysis of serum samples from 202 healthy individuals collected between 2004 and 2010 revealed that anti-stem antibodies of group 1 specificity is found in 84% of the population ; howeverEllebedy and colleagues  found thAlthough HA stem is an excellent candidate for the development of universal vaccines, anti-HA stem titers in human correlates only with reduced viral shedding but do not predict the severity of influenza infection , 57. A rThe globular head of the HA is target for most of the neutralizing antibodies, which prevents the viral entry by blocking the binding of RBS to sialic acid residues on host cell membrane . RBS is Apart from RBS, broadly neutralizing antibodies have been identified against other conserved sites on HA head . An antide novo protein design tools, we will be able to create effective immune-focusing antigens  are full-length HA with stem derived from human viruses and globular head from distinct, exotic HAs. Based on this concept, repeated immunization with cHA with head from different flu subtype and same H1 HA stem induced high titers of stem-reactive antibodies against homologous and heterologous viruses . SeveralN-linked glycosylation on HA has been known to stabilize HA and shield virus from host immune response . CreatinKanekiyo et al. used a ferritin nanoparticle displaying a repetitive array of HA hyper variable receptor binding domains (RBD from different H1N1 strains). Using this, they could subvert B cell response from strain-specific immunodominant epitopes to conserved, shared epitopes. Since cross-reactive B cells can recognize conserved epitope from an heterogenous array of RBD, these cells get preferentially activated due to avidity advantage and could selectively activate pre-existing cross-reactive Bmem. One of the antibodies generated after immunization, 441D6, binds to a conserved epitope, which lies opposite to RBS and protects against H1N1 strains spanning from 1977 to 2009 , 114. AnM2 is an ion channel with its ectodomain (M2e) exposed on virion and infected cell surfaces . M2e is in vivo (IAV NA as vaccine target has been neglected for decades, despite early discovery of potent anti-viral activity of NA antibodies . Even moin vivo . Still, in vivo –141. By Bioengineering and design of epitope-focused immunogens is proceeding at an incredible speed in influenza and other fields. Several promising immunogens are now in clinical trials and, hopefully, will be available to the public soon, as long-lasting universal vaccines. It is, however, crucial to understand more about the basics of B cell responses to interpret results and inform on vaccination policies.Introduction of pandemic H1N1 2009 virus showed that most individuals, with low serological anti-stem antibodies, were able to mount a stem-directed response, but repeated vaccinations skewed the immune response back to the immunodominant head . It willFinally, but not less important, new vaccines platforms are constantly being tested. RNA-based vaccines have shown exciting results when expressing influenza proteins, at least in animals [reviewed in ]. Some oWe are just entering an exciting season of clinical trials, and while expectations are really high, we should not be discouraged if some of the early attempts fail but rather persevere in the quest for a universal and long-lasting vaccine.NM and DA researched the literature and wrote the manuscript.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.


The use of testicular in preference over ejaculated sperm for intracytoplasmic sperm injection (ICSI) has gained increased attention due to reports of better pregnancy outcomes using testicular sperm for cases of infertility associated with high sperm DNA fragmentation (SDF) . Indeed“Infertile couples undergoing ICSI, whose male partners have elevated SDF levels in the neat ejaculate, should be offered testicular sperm in the next ICSI cycle, provided SDF is persistent after treatment of the underlying condition, or the clinical scenario does not allow treatment”. There are three essential, evidence-based premises supporting this clinical approach, which I will discuss in the next paragraphs.The matter concerned has been subjected to opinionated debate as Testi-ICSI represents a paradigm shift in clinical practice , 3-5. I in vitro fertilization (IVF) and ICSI pregnancy outcomes but also contributes to pregnancy loss. In fact, results from the most recent and largest systematic review and meta-analysis about the impact of SDF on assisted reproductive technology (ART), which pooled data from 70 studies and over 17,000 IVF and ICSI cycles, indicate that SDF reduces the probability of a successful pregnancy following ART  . LikewisSecond, testicular sperm have lower DNA fragmentation than ejaculated counterparts in men with elevated SDF in the neat ejaculate. This conclusion derives from a recent systematic review and meta-analysis, including five studies and 143 patients who served as their control, that is, SDF was measured in ejaculated and testicular specimens obtained from the same men . Four stA study from our group, included in the meta-analysis mentioned above, compared DNA fragmentation rates between ejaculated and testicular sperm in 81 men with idiopathic oligozoospermia and elevated DFI . In our One of the main reasons why SDF is higher in semen than testis relates to the susceptibility of sperm chromatin to oxidative attack, particularly during epididymis transit . Apoptos2=34%, P<0.0001) and the results were conservative in subgroup analyses including couples with ICSI failure  or first ICSI comers . Furthermore, the OR of a live birth also favored testicular sperm . Importantly, the odds of a miscarriage were reduced by approximately 67% overall using testicular sperm   . The conA prospective, observational cohort study from our group (evidence level 2b), which had a substantial weight (59%) in the meta-analysis discussed above, included 147 infertile couples . The menAlthough the current data supporting testicular sperm for selected non-azoospermic infertile men is reassuring, it is important to recognize the existence of gaps in knowledge and the risks of sperm retrieval, as discussed in detail elsewhere . BrieflyIn my practice, I request SDF testing to selected ART candidates and recommend treatment of the underlying conditions associated with SDF . VaricocWe rely on testicular sperm instead of laboratory methods to select specimens with lower SDF levels because it allows for sperm acquisition before transiting through the epididymis, which is when SDF is thought to be acquired. Also, it has been shown that Testi-ICSI provides higher LBR when compared with laboratory methods such as physiological intracytoplasmic sperm injection (PICSI) and intracytoplasmic morphologically selected sperm injection (IMSI) in couples with high SDF in semen . In a 20Lastly, as for the health of offspring resulting from Testi-ICSI in cases of high SDF, there is lack of published data. However, reports of ICSI using testicular sperm in azoospermia have been overall reassuring, as no major differences are noted in the short-term neonatal outcomes and congenital malformation rates among children from fathers with nonobstructive azoospermia or obstructive azoospermia , 28.In conclusion, I first presented evidence confirming that SDF negatively impacts ART pregnancy outcomes and is associated with pregnancy loss. Then, I provided data to substantiate the premise that SDF is lower in testicular than ejaculated sperm among infertile men with high SDF in semen. Lastly, I summarized the evidence supporting the proposition from which the use of testicular in preference over ejaculated sperm (therefore with lower SDF) is associated with improved ICSI pregnancy outcomes in couples whose male partners have high SDF in semen. These evidence-based premises make the argument stated at the beginning of this article “Infertile couples with ICSI failure should be offered Testi-ICSI if male partners have high SDF in the neat ejaculate” irrefutable. Therefore, Testi-ICSI should be considered in the treatment plan of infertile couples undergoing ICSI when the following conditions are met (i) Presence of high SDF levels in neat ejaculate, measured by a reliable assay with a validated threshold, and (ii) Persistence of elevated SDF levels despite treatment of the underlying condition causing SDF (if correctable). A failed ICSI cycle using ejaculated sperm with no other obvious reasons explaining that failure should reinforce consideration for the use of Testi-ICSI, provided the conditions mentioned above are met.Expanded from an invited talk by the author delivered at the American Society for Reproductive Medicine (ASRM) Annual Meeting, San Antonio, USA, October 2017.