The effects of noninvasive neurostimulation on brain structure and function in chronic poststroke aphasia are poorly understood. We investigated the effects of intermittent theta burst stimulation (iTBS) applied to residual language-responsive cortex in chronic patients using functional and anatomical MRI data acquired before and after iTBS. Lateralization index (LI) analyses, along with comparisons of inferior frontal gyrus (IFG) activation and connectivity during covert verb generation, were used to assess changes in cortical language function. Voxel-based morphometry (VBM) was used to assess effects on regional grey matter (GM). LI analyses revealed a leftward shift in IFG activity after treatment. While left IFG activation increased, right IFG activation decreased. Changes in right to left IFG connectivity during covert verb generation also decreased after iTBS. Behavioral correlations revealed a negative relationship between changes in right IFG activation and improvements in fluency. While anatomical analyses did not reveal statistically significant changes in grey matter volume, the fMRI results provide evidence for changes in right and left IFG function after iTBS. The negative relationship between post-iTBS changes in right IFG activity during covert verb generation and improvements in fluency suggests that iTBS applied to residual left-hemispheric language areas may reduce contralateral responses related to language production and facilitate recruitment of residual language areas after stroke. Strokes of the left middle cerebral artery (LMCA) territory often lead to impairments in language function that are collectively referred to as aphasias . LanguagFunctional neuroimaging studies indicate that the recovery of language abilities after LMCA stroke involves the restoration of language-related processing in the remaining tissues near affected language areas as well as the compensatory recruitment of unaffected areas for language-related processing –14. WhilStudies investigating how changes in cortical function relate to language recovery following stroke provide strong evidence indicating that the preservation and/or restoration of language-related processing in the residual left inferior frontal gyrus (IFG), a region that has been strongly implicated in various language processes such as word processing and word generation –26, is sTechniques such as transcranial magnetic stimulation (TMS) enable the noninvasive manipulation of cortical excitability in specific parts of cortex and may provide a means for facilitating beneficial cortical plasticity in patients with poststroke aphasia , 29. ExpExcitatory stimulation protocols are typically applied to the residual left IFG in order to facilitate language-related processing –36. In cA previous behavioral and functional MRI (fMRI) study conducted by our laboratory found that after 10 sessions of iTBS applied to residual language-responsive left frontal cortex identified with a semantic decision/tone decision task, patients with chronic poststroke aphasia showed significant improvements in word generation as well as changes in fMRI responses during a semantic decision task that included a significant leftward shift in the lateralization of activity in the IFG . In addiHere, we first analyzed the pre-/postintervention fMRI data to assess whether or not iTBS might have similar effects on fMRI responses elicited by a covert verb generation (VG) paradigm that was not used to define iTBS targets. Notably, while both the semantic decision paradigm used in our previous study and the VG paradigm used in the current study reliably evoke strong responses in the left IFG in healthy individuals  and in pEight prospectively identified patients  with chronic aphasia resulting from LMCA stroke were recruited as described previously , 41. TheDetailed descriptions of all iTBS and neuronavigation protocols performed on these patients can be found in our previous publication . Briefly∗-weighted blood oxygen-level dependent (BOLD) volumes  were obtained at both pretreatment and posttreatment sessions.MRI data were acquired before and after the treatment sessions. The functional and anatomical MRI data presented in this study were acquired using a Varian 4 Tesla Unity INOVA whole body MRI/MRS scanner . For each patient, a high-resolution T1-weighted 3D-MDEFT (Modified Driven Equilibrium Fourier Transform) anatomical volume  and T2FMRI data were collected while patients performed an alternating block-design covert verb generation (VG) task that consisted of alternating 30 s blocks of an active condition involving silent verb generation in response to binaurally presented nouns and a control condition involving bilateral sequential finger tapping (FT) in response to a frequency modulated tone centered on 400 Hz that was modulated by 25% every 5 s. This task was chosen because previous studies indicate that it reliably produces left-lateralized activation patterns  and becaAll MRI data were preprocessed using MATLAB scripts implementing functions from the most recent release version of Statistical Parametric Mapping  running in MATLAB r2014B . All statistical analyses were performed using statistical functions provided in MATLAB.Functional MRI data from the baseline and follow-up scans were slice-time corrected, realigned and resliced, and coregistered to the structural image obtained during the same scan. Deformation fields containing the deformation differences between across-session average anatomical volume and the anatomical scan from each session were used to warp the coregistered functional volumes to the across-session average anatomical volume. The average anatomical scan was then normalized to Montreal Neurological Institute (MNI) template using unified normalization-segmentation as implemented in the New Segment tool in SPM. The deformation parameters obtained from the warping of the anatomical volume were used to normalize the functional volumes to MNI template space. The functional volumes were resampled to 2 × 2 × 2 millimeter isometric voxels and spatially smoothed with a 6-millimeter full-width half maximum (FWHM) Gaussian kernel. Functional volumes in which participants moved more than 0.5 mm in one frame (3 s) were replaced with a volume interpolated from adjacent time points. Volumes were to be rejected if they contained >3 mm of motion, but no volumes met criteria for rejection.Individual patient lesion delineations were created from the pre-iTBS anatomical scans using an automated voxel-based Bayesian classification algorithm developed by our lab and implemented in the lesion_gnb toolbox for SPM12 . The resAnatomical data utilized in the voxel-based morphometry (VBM) analysis were preprocessed according to a recently described longitudinal preprocessing pipeline for VBM analyses . First, Functional MRI activity related to the covert verb generation task was quantified by contrasting the active condition blocks (VG) against the control condition blocks (FT). For each patient, the fMRI data were fit to a general linear model (GLM)  where eahttp://marsbar.sourceforge.net/), and ROIs in the left and right IFG were defined using peak activation coordinates obtained from an independent analysis of activation related to covert verb generation in healthy individuals [x = −50, y = 16, and z = 16) and on mirrored coordinates in the right IFG . Only two patients (P1 and P4) had overlap between the left IFG ROI and their lesion delineation. P1's lesion encompassed nearly the entire left lateral prefrontal cortex and the left IFG ROI fell directly onto the perilesional rim, resulting in 100% lesion overlap with the left IFG ROI. P4's lesion was primarily localized to the left ventral IFG and the ROI overlapped by 60.2% with the perilesional rim. Nonetheless, for both patients, the 1st principal component of the signal extracted from the ROI showed phasic responses consistent with the design of the task. While anatomical overlap was noted, functional analyses indicated BOLD signal changes aligned with the box-car function of the fMRI task design indicating that the fMRI responses reflected the responses of perilesional cortex rather than CSF. Additionally, signal from the left IFG ROI was more strongly correlated with signal from the right IFG ROI than with the CSF signal, and this was only marginally influenced by partialling out variability accounted for by the CSF signal  . Nonetheless, control analyses indicated that excluding these patients did not substantially change the statistical significance of results for any analysis involving the left IFG ROI, indicating that the presence of lesion-ROI overlaps for these patients did not strongly influence the outcome of the ROI analyses.FMRI data were first analyzed using a region of interest (ROI) approach in order to directly test our hypotheses regarding changes in IFG activity and connectivity between pre- and post-iTBS sessions. ROI masks were created using the marsbar toolbox for SPM ; this method does not substantially increase susceptibility to false positives [To test our hypothesis about whether patients showed changes in fMRI activity during covert verb generation in the left and right IFG ROIs after iTBS, we extracted the mean parameter estimates for the active versus control condition contrast and compared the estimates obtained for each ROI between the pre- and post-iTBS sessions. This analysis provided information about the mean level of activity in the IFG ROIs for each session, with positive values indicating stronger activation during the active condition and negative values indicating stronger activation during the control condition. To test our hypothesis that patients would show more strongly left-lateralized IFG activation following iTBS, laterality index (LI) analyses were performed to quantify the lateralization of activity related to covert verb generation. For each patient, changes in LI were evaluated using an adaptive threshold determination approach . LI valuositives . The masLI=∑activositives .To test our hypothesis regarding the effects of iTBS on interhemispheric connectivity, we conducted a generalized psychophysiological interaction (gPPI) analysis using the gPPI toolbox for SPM . gPPI ent-tests. Correlations between changes in fMRI measures of IFG function and behavioral measures were assessed using linear correlation analyses. Multiple-comparisons correction for all ROI-driven comparisons between pre-iTBS and post-iTBS scans was performed using the Benjamini-Hochberg procedure to control the false-discovery rate (FDR) at 0.05 [P values presented are FDR-adjusted. Exploratory whole-brain GLM and gPPI analyses were also performed in order to provide a more thorough characterization of functional MRI measures of activity and right IFG connectivity related to the VG task at the pre-iTBS and post-iTBS sessions. Statistical tests for these analyses were performed using dependent samples t-contrasts. Exploratory and ad hoc partial correlational analyses were performed to further characterize the data and are presented with uncorrected P values.All between-session comparisons were tested for statistical significance using two-tailed dependent samples  at 0.05 , 68, andt-contrast that also included each patient's lesion volume as a nuisance covariate. Changes in GM volume were assessed at the whole-brain level using dependent samples t-contrasts.VBM is a technique that allows for the measurement of grey matter (GM) volume in T1-weighted MRI data , 69. Hert-test comparing pre-iTBS and post-iTBS evaluations did not reveal a significant change in noun recognition performance . These results are consistent with previous studies that have reported good out-of-scanner noun recognition performance on this task in patients with poststroke aphasia [Analyses evaluating performance on the out-of-scanner forced-choice noun recognition task revealed good performance for both pre-iTBS  and post-iTBS  sessions. Dependent samples  aphasia , 70.P < 0.05) improvement on the Semantic Fluency Test, statistically nonsignificant (at P < 0.05) improvements in performance on the Boston Naming Test , and a statistically nonsignificant (at P < 0.05) decrease in performance on the Controlled Oral Word Association Test . Since  a priori in order to avoid the introduction of bias by defining ROIs based on the GLM results. Our analyses revealed increased activation magnitudes in the left IFG  and decreased activation magnitudes in right IFG  related to covert verb generation after iTBS treatment. Left and right IFG activation magnitudes for each patient are shown in Figures t7 = 3.46, FDR P = 0.02; mean change = 0.48, SEM = 0.14). LI estimates for each patient at pre-iTBS and post-iTBS sessions are shown in To test our hypotheses regarding the effects of iTBS on the magnitudes of activity in left and right IFG during covert verb generation, we compared activation magnitudes at each ROI between pre- and post-iTBS sessions. It is worth noting that the ROIs used for these analyses were chosenP < 0.001, uncorrected) increases in activity were observed in the left IFG pars opercularis , the right thalamus , and the right cerebellum VI , and the most reliable  decreases in activity were observed in the right cerebellum crus 2 , the right cerebellum VIII , and the right inferior temporal gyrus .To allow for a more thorough characterization of the data, whole-brain GLM analyses were also performed. Although no regions showed significant effects at a whole-brain FDR-corrected threshold of 0.05, the uncorrected statistical maps provide evidence for increased responses related to covert verb generation in left-hemisphere frontal, temporal, and parietal regions after iTBS . While nt7 = −2.97; FDR P = 0.03; mean change = −0.24, SE = 0.09). The gPPI estimates for the active condition contrast for each patient are shown in To test our hypothesis regarding the effects of iTBS on effective connectivity between the right and left IFG, we compared gPPI estimates between the right IFG seed region and the left IFG target region between pre- and post-iTBS sessions. It is important to note that gPPI estimates reflect the magnitude of condition-dependent changes in the relationship between activity in the seed region and activity in the target region . Thus, tSince the gPPI measurement quantifies differences in the relationship between activity in the seed region (R IFG) and the target region (L IFG) that are moderated by task condition (VG-FT), a reduction in the gPPI estimate between right IFG and left IFG would indicate that the effect of right IFG activity on left IFG activity for covert verb generation relative to finger tapping was reduced after iTBS. Patients showed a small but positive mean effect of covert verb generation on connectivity between the right IFG and left IFG pre-iTBS, indicating that right IFG activity was more positively associated with left IFG activity during covert verb generation than during finger tapping. In contrast, patients showed a negative mean effect of covert verb generation on connectivity between the right IFG and left IFG post-iTBS, indicating that right IFG activity was more negatively associated with left IFG activity during covert verb generation than during finger tapping. Thus, the direction of the effect of task condition on the relationship between right IFG activity and left IFG activity changed between pre-iTBS and post-iTBS sessions, with right IFG activity being more negatively associated with left IFG activity during covert verb generation than during finger tapping.P < 0.001, uncorrected) reductions in right IFG connectivity associated with the VG task were observed in the right middle temporal gyrus , the right superior frontal gyrus , the left IFG pars opercularis , the right postcentral gyrus , the left lingual gyrus , the right cerebellum VI , the left caudate , and the left temporal pole . Interestingly, comparable  increases in right IFG connectivity associated with the VG task were not observed after iTBS.To allow for a more thorough characterization of the data, whole-brain gPPI analyses were also performed. Although no regions showed significant effects at a whole-brain FDR-corrected threshold of 0.05, the uncorrected statistical maps provide evidence for reduced connectivity between the right IFG and left-hemisphere frontal, temporal, and parietal regions after iTBS . While nr = 0.5, P = 0.2) and changes in connectivity  and weak but nonsignificant correlations between total lesion volume and right IFG activity . Thus, partial linear correlations were computed to investigate the relationship between the changes in functional MRI measurements of IFG function during covert verb generation and changes in performance on the Semantic Fluency Test following iTBS that were not attributable to total lesion volume. These analyses did not reveal significant correlations between overall changes in LI and changes in performance , changes in the magnitude of left IFG activity and changes in performance , or changes in the effects of covert verb generation on interhemispheric connectivity and changes performance . These analyses did reveal a strong negative correlation between changes in the magnitude of right IFG activity and changes in performance , indicating that decreases in right IFG activity during covert verb generation between pre/post-iTBS sessions were associated with concurrent improvements on the Semantic Fluency Test.Prior to assessing behavioral correlations with fMRI measures of IFG function, correlations between total lesion volume and each measure were assessed. This revealed moderate but nonsignificant correlations between total lesion volume and changes in L IFG activity . Second, we addressed the question of whether or not the effects of iTBS on left and right IFG activation magnitudes were correlated with the effects of covert verb generation on interhemispheric connectivity prior to iTBS treatment. This revealed a positive correlation between pre-iTBS effects of verb generation on interhemispheric connectivity and changes in left IFG activation magnitude after iTBS treatment . A nonsignificant negative correlation was found between pre-iTBS connectivity and changes in right IFG activation magnitude after iTBS .First, we addressed the question of whether or not changes in left IFG activity after iTBS were correlated with changes in right IFG activity after iTBS while controlling for total lesion volume. This revealed a nonsignificant negative correlation between post-iTBS changes in left and right IFG activity  increases in GM volume occurred in the left medial orbital gyrus  and in the left lingual gyrus , and the most reliable  decreases in GM volume occurred in the right superior frontal gyrus  and in the right IFG pars opercularis .VBM was used to test our hypothesis that patients would show changes in GM volume between pre- and post-iTBS sessions. An initial analysis using a voxelwise FDR threshold of 0.05 did not reveal any effects of iTBS on GM volume. An evaluation of the unthresholded statistical maps  revealedGrowing evidence supports the use of techniques that utilize the noninvasive modulation of cortical excitability to improve language functions in patients poststroke aphasia , 71–73. The rerecruitment of residual left-hemisphere cortex for language processes is likely an important factor for the optimal long-term recovery of language functions following LMCA stroke , 21, 23.A speculative explanation for the observed effects is that the changes in left versus right IFG activity and connectivity during covert verb generation reflect the reinstatement of balanced inhibitory interactions between left and right IFC , 75. DisAdditionally, results from our post hoc analyses indicated that the effects of iTBS on left and right IFG function had opposite relationships with the pre-iTBS strength of right IFG to left IFG connectivity during covert verb generation. While exploratory and requiring additional validation, these results have important implications, as they suggest that preexisting interhemispheric dynamics contribute to the effects of iTBS on the function of both the stimulated and unstimulated hemispheres. This interpretation is consistent with results from a recent study that indicated that the preservation of frontal white matter tracts, specifically the left arcuate fasciculus, explains substantial interindividual variability in behavioral improvements following cathodal TDCS applied to right IFG in patients with left IFG lesions . Our resNevertheless, it is pertinent to address the question of how such large-scale changes in cortical function might result from the passive stimulation of residual left-hemisphere areas. After stroke, the loss of large-scale neural populations is thought to result in an acute breakdown of function in large-scale cortical networks that enable complex cognitive functions such as language  and atteWhile the combination of iTBS with active language therapy might also be expected to lead to an enhancement of beneficial neuroplasticity, the expectation that passive stimulation can lead to changes in task-driven responses and connectivity is not unfounded. Indeed, the cortical/subcortical networks that underlie cognitive and behavioral functions such as language and attention maintain ongoing interregional signaling even when tasks are not being performed –83. ImpoThe present study has limitations that must be acknowledged and considered in interpreting the results and in designing future studies. Primarily, the lack of a sham-stimulation group precludes the ability to make definitive statements about whether the observed effects are specific to iTBS treatment. It is worth noting that activation patterns during the covert verb generation task have been found to be remarkably consistent across time in patients with chronic poststroke aphasia , and theWhile sham-controlled studies investigating the effects of neurostimulation on language function in patients with chronic poststroke aphasia have consistently reported behavioral improvements that are specific to real stimulation , 73, 85,P value thresholds for the whole-brain analyses, the results from these analyses should be interpreted with caution. As such, we have refrained from drawing strong conclusions about the effects of iTBS on GM volume or on activity/connectivity beyond those examined with our ROI analyses.A second limitation to this study is the relatively small sample size. Although some would argue that this property makes the observed significant effects more compelling since the likelihood of finding significant-but-trivial effects increases with sample size, our limited sample likely also led to the obscuration of real effects due to relatively low power . Indeed,While the whole-brain results are exploratory and thus should not be used to draw strong conclusions about the effects of iTBS, they do merit discussion. Regarding the observed effects of iTBS on regional GM volume, it would not be surprising if iTBS did have an effect on GM morphology in these patients, as detectable changes following rTMS have been reported after in as little as five days by studies investigating the effects of rTMS on cortical morphology in healthy individuals . It is aNCT01512264) in order to better clarify the neuroplastic effects of iTBS in this population.In conclusion, we investigated the effects of iTBS applied to residual language-responsive cortex in the left hemisphere on MRI measurements of cortical function and structure in eight patients with chronic poststroke aphasia. We found that iTBS was associated with increased left-lateralization of IFG activity during covert verb generation. Changes in lateralization were characterized by increases in left IFG activation magnitudes and decreases in right IFG activation magnitudes that presented as an overall shift in the lateralization of IFG activity during covert verb generation. iTBS also led to reduced right to left IFG connectivity during covert verb generation, consistent with our interpretation that the effects of iTBS are related in part to changes in context-dependent interhemispheric interactions. Interestingly, our post hoc analyses suggest that the effects of iTBS on left and right IFG function were negatively correlated across patients (increased left IFG activity was associated with decreased right IFG activity), and the changes in left versus right IFG responses had opposite relationships to pre-iTBS levels of right IFG to left IFG connectivity during covert verb generation. These data provide insights into the neuroplastic changes associated with iTBS applied to residual left-hemisphere language areas in the treatment of chronic poststroke aphasia and provide support for future research in this area. Randomized, blinded, and sham-controlled studies in a larger sample of patients are necessary and are currently in progress . In addition, correlational analyses are provided to show that for both patients, the left IFG ROI time-series was more strongly correlated with the right IFG ROI than with the CSF time-series, and that the right-left IFG correlation was only marginally affected by removal of the CSF signal. These analyses demonstrate that the ROI-lesion overlap was unlikely to introduce artifactual effects into the left IFG signal.


Clinicians are faced with unprecedented opportunities to identify the genetic aetiologies of hitherto molecularly uncharacterised conditions via the use of high-throughput sequencing. Access to genomic technology and resultant data is no longer limited to clinicians, geneticists and bioinformaticians, however; ongoing commercialisation gives patients themselves ever greater access to sequencing services. We report an increasingly common medical scenario by describing two neuromuscular patients—a mother and adult son—whose consumer access to whole-genome sequencing affected their diagnostic journey.COL6A1, COL6A2 and COL6A3. This targeted approach revealed a heterozygous causative variant in COL6A3 ), shared by both individuals, that was not flagged by the interpretation of the whole-genome sequencing data.Whole-genome sequencing initiated by the patients—to predict their risk of common diseases—revealed that they share several variants potentially relevant to neuromuscular diseases, which initially sidetracked diagnostic efforts. Since eventual clinical reassessment, including muscle imaging, pointed towards Bethlem myopathy, a collagen VI-related myopathy, we pursued Sanger sequencing of This report highlights the essential interplay of clinical and genomic expertise in realising the potential of high-throughput sequencing. In an era when patients themselves may bring their own data to the table, definitively identifying clinically significant genomic variants will require close collaboration among clinicians, geneticists and bioinformaticians. Advantages of whole-genome sequencing over targeted, exon capture approaches in genetically heterogeneous Mendelian disorders were reported by Lupski The successes of whole-genome sequencing reflect the combined interpretive skills of geneticists, bioinformaticians and clinicians working in close collaboration. Indeed, this essential interplay of complementary genomic and clinical expertise is epitomised by the recent identification of new genes in hitherto molecularly uncharacterised conditions. Importantly, patients’ own independent access to genomic technology may catalyse such interaction but may also steer the diagnostic journey from its conventional path, with varied results. The following case highlights this contemporary medical scenario and serves as an instructive tale.The proband, a 33-year-old male, presented to our (RDSP and MGH) neuromuscular centre in London in 2004. He was never able to run, and he had developed slowly progressive proximal weakness beginning in his teenage years. Examination revealed bilateral elbow contractures, Achilles tendon tightness and proximal muscle weakness -grade 4/5). Deep tendon reflexes were present. Creatine kinase was 354 IU (reference range: <150 IU). Muscle biopsy revealed evidence of muscle fibre necrosis and regeneration, consistent with an underlying dystrophy A–D.22The patient's mother reported a similar history, albeit with a later onset, with proximal muscle weakness noted around age 30 years and progressing to dependence on a cane for walking by age 60 years. Muscle biopsy was reported as dystrophic-appearing but was not available for review.No other family members were noted to have similar features. The family was counselled that their condition was likely a form of autosomal dominant muscular dystrophy, with Bethlem myopathy and Emery-Dreifuss muscular dystrophy included in the differential diagnosis. Further investigations were recommended; however, the family did not return to our clinic for the ensuing 5 years.COL6A1 that is modestly common  in the general population and could not alone plausibly underlie the phenotype.When the family returned in 2010, they brought an electronic data storage device with their annotated whole-genome sequence data, obtained—in order to predict their risk of common diseases—through sequencing contracted through our  genome interpretation company in Boston. Analysis of the kindred genome data flagged protein-altering non-reference variants in 22 neuromuscular disease-implicated genes that were called in the proband and his mother but not the proband's unaffected father or wife (unrelated), who also underwent whole-genome sequencing. The strongest such candidate, however, was a heterozygous missense variant in SGCA and SGCG) were each called with a heterozygous protein-altering variant shared by the proband and his mother. Variants in these genes have been recessively (or within one gene compound heterozygously) implicated in sarcoglycanopathies, but we knew of no report tracing disease to interaction between heterozygous variants in distinct sarcoglycan genes. Moreover, the clinical phenotype of the patients was not consistent with phenotypes associated with sarcoglycanopathies. As such, the clinical and genome interpretation teams began a close dialogue, to best integrate the initially inconclusive genomic findings with detailed clinical findings, for further diagnostic insight.Two other neuromuscular disease-implicated genes .6–8In order to further elucidate the patients’ pattern of muscle involvement and decipher the potential relevance of genetic variants of unknown significance, we  recommended lower extremity muscle MRI be performed, since it can be instrumental in narrowing the differential diagnosis and pointing towards particular genetic aetiologies.COL6A1, COL6A2 and COL6A3 in a diagnostic laboratory in London. Strikingly, the targeted sequencing and interpretation flagged a heterozygous c.6365G>T  novel variant in exon 20 of COL6A3 in the proband and his mother. Such variants, in the triple helical domain of the collagen VI protein, are known to result in aberrant formation of the extracellular matrix protein collagen VI, resulting in a spectrum of collagen VI-related myopathies ranging from Bethlem myopathy to the more severe and allelic condition Ullrich congenital muscular dystrophy.11Given this muscle MRI pattern strongly evocative of Bethlem myopathy and consistent with our clinical impression, and since causative variant(s) in the collagen 6 genes were not flagged by the interpretation of the whole genome sequencing data, we pursued follow-up long-read (Sanger) sequencing of et alsupplementary figure S1A,B), thus providing further evidence of collagen VI deficiency. Sequencing of complementary DNA, extracted from skin fibroblasts, confirmed the presence of the COL6A3 glycine-altering variant called in the proband and his mother's genomic DNA, which did not result in abnormal splicing.Skin biopsies performed in the proband and his mother for dermal fibroblast collagen VI immunocytochemistry and flow cytometry studies  were excluded. Counselling regarding the natural history of Bethlem myopathy, its autosomal dominant inheritance pattern in this family and the associated risk of recurrence were provided to the proband and his mother in the setting of our (RDSP and MGH) neuromuscular centre. In contrast, when the family received the annotated data of their independently contracted whole-genome sequencing and interpretation, they did not have genetic counselling.COL6A3 variant was detected, but not interpretively flagged, due to the inadequacy of our  pipeline (at the time) for predicting functional effects of variants in whole-genome analysis. Specifically, our whole-genome analysis pipeline relied on data published in conjunction with the SIFT (Sorting Intolerant From Tolerant) algorithm to identify, and optionally functionally prioritise, missense variants. At the time, SIFT annotated the COL6A3 transcript in question on the wrong strand, and our pipeline did not yet correct, or flag, such discrepancies. By contrast, the conventional pipeline used to interpret targeted sequencing of collagen genes successfully flagged the variant in question as functionally relevant/potentially deleterious.Re-analysis of the whole-genome sequencing data confirmed that the causative COL6A3 variant. Instead, this variant was identified by Sanger sequencing of the collagen 6 genes, motivated by muscle MRI findings, per the conventional diagnostic pathway. While whole-genome reference data have since improved to better flag potentially harmful variants in COL6A3, whole-genome sequencing and interpretation will likely remain ‘blind’ to some variants that targeted methods identify more reliably. Such whole-genome shortcomings are inherent to methods of short-read sequencing  and interpretation . Conventional locus-specific databases, such as those available through the Leiden Open Variation Database (LOVD), may contain more reliable data on particular genes than do sources casting a broader net on our genomes. Such locus-specific databases cover only particular genes, and use sundry formats that defy easy integration for whole-genome interpretation, but they will likely remain a cornerstone of interpretation in difficult cases.The diagnostic journey outlined here is provocative for several reasons. First of all, whole-genome sequencing independently pursued by our patients did not uncover the causative Second, this case highlights muscle MRI as a valuable diagnostic tool for Bethlem myopathy, for which it reportedly yielded 96% diagnostic specificity in a study of patients with Bethlem myopathy and other muscular dystrophies presenting with rigid spine.13Third and most importantly, the diagnostic trajectory described here highlights the indispensable role of clinical expertise in the era of genomic medicine. Clinicians, geneticists and bioinformaticians together face the challenge of wading through an immense quantity of data from patient genomes—a challenge compounded if any of them tries to navigate these waters in isolation.The commercialisation of high-throughput sequencing alters the landscape of information available to patients, yielding vast data but no easy means to definitively interpret it. Notably, clinician-initiated high-throughput sequencing is now widely available, but always requires formal ethics oversight and, if for research, express institutional approval. By comparison, restrictions on consumer-initiated sequencing vary more widely and in some jurisdictions allow detailed genomic data to be directly available to patients and their relatives—data that will often then be presented to a clinician for review, as in this case. Given the rapid decline of the cost of whole-genome sequencing, many clinicians may soon confront the scenario presented here. Indeed, cost is no longer the limiting factor in whole-genome sequencing, which in 2001 cost ∼US$100 000 000 but in 2011 fell below US$10 000 .The greatest hurdle to the use of high-throughput sequencing in clinical practice may, in fact, be time. Genomicists, bioinformaticians and clinicians worldwide must work together, carefully and patiently scrutinising immense quantities of genomic data, on a joint quest to identify variants of clinical significance. Without coordinated, dedicated efforts, the course of genomic medicine risks Dickens’ admonition: ‘we had everything before us, we had nothing before us.’15


Compartmentalization of intracellular membrane is a marked characteristic during the pathogenesis of many viruses, which provides a microenvironment for virus to evade the host RNA interference defense response on one hand, and confines various host factors as well as metabolites in a stable space so that they can act in concert to complete virus replication cycle . Recentlet al., for the first time, resolved the 3D architecture of VRCs, which shows the outer mitochondrial membrane-invaginated 50 nm vesicles (spherules) in the flock house virus (FHV)-infected Drosophila cells [et al demonstrated that the enrichment of phosphatidylethanolamine at the membrane-bound VRCs is an essential step for a (+)RNA virus replication [3D analysis of various virus replication complexes (VRCs) revealed that, albeit with their own distinct traits, structural similarities are present among different VRCs, suggesting viruses might adopt conserved strategies to build the replication factory during long-term evolution. For example, Kopek la cells . Typicalla cells . The morla cells . It is ilication . Thus, eIt should also be pointed out that one or several virus-encoded proteins usually act as key triggers of VRCs morphogenesis. Sometimes expression of a single viral protein alone in the cells could form identical cytopathological structures with that of corresponding viral infections . Hence, Taken together, the 3D views of viral replication factories provide new insight into virus-host interaction and should greatly facilitate the discovery of potential targets for antiviral therapeutic intervention.


Background: Sinus of Valsalva-right ventricle fistula is a recognized but very rare complication after surgical repair of subaortic ventricular septal defect. Surgical repair with cardiopulmonary bypass and percutaneous transcatheter closure guided by x-ray has been the traditional treatment for fistula of sinus of Valsalva. Case Presentation: Recently, we have used a novel approach, that avoids the need for either secondary open surgical repair or radiation exposure; that is, minimally invasive transthoracic device closure guided by transesophageal echocardiography to occlude an acquired sinus of Valsalva-right ventricle fistula in a 4-year-old patient. Conclusion: To our knowledge, there have been no prior cases reported of this technique applied to close an acquired sinus of Valsalva-right ventricle fistula. This report aims to provide a detailed description of the procedure. Cardiopulmonary bypass and percutaneous transcatheter closure are the 2 generally used methods to treat fistula of sinus of Valsalva. Here we present a novel method -minimally invasive transthoracic device closure guided by transesophageal echocardiography (TEE) - that we used to occlude an acquired sinus of Valsalva-right ventricle fistula after surgical repair of ventricular septal defect (VSD) in a 4-year-old patient.Sinus of Valsalva-right ventricle fistula is a recognized but very rare complication after surgical repair of subaortic ventricular septal defectThe 4-year-old girl underwent subaortic VSD repair surgery in our hospital on October 24, 2011 when she was 2 years old. Ten days after surgery, transthoracic echocardiography showed that the VSD was properly repaired but there was a fistula of right sinus of Valsalva (RSOV) to the right ventricle (RV). With the fistula’s diameter of 1.5 mm, color Doppler showed a continuous shunt from RSOV to RV without aortic regurgitation (AR), and physical examination showed a new grade 3/6 continuous murmur at the left sternal border. Since no rupture of sinus of Valsalva was found in the preoperative echocardiography ordetected during surgery, it was believed that this fistula was a postsurgical complication. Because the patient was asymptomatic and the fistula was small, her parents decided to observe her for progression over a period of time. During 21 months of observation, transthoracic echocardiography (once every 3 months) showed that the fistula’s diameter increased from 1.5 mm to 2.5 mm with a high-velocity (4.5 m/s) continuous Doppler flow , B, C. M Although the initial treatment recommendation was surgery, the patient’s parents were more inclined to avoid reopening the heart and using cardiopulmonary bypass and eliminating radiation exposure during percutaneous closure. With experience in closed VSDs using minimally invasive transthoracic device closure guided by TEE, we explored the feasibility of adopting this technique to close the fistula. After the potential risks were explained in detail, the parents gave their informed consent for occlusion of the fistula of RSOV under TEE guidance.  Under general anesthesia, TEE confirmed the diagnosis of fistula of the RSOV to RV  with a fAfter deployment, TEE showed that the occluder had a stable position with abolition of the shunt, and no AR occurred . No othe Three months after intervention, the patient was in good condition with no cardiac murmurs on examination.Transthoracic echocardiography revealed satisfactory position of the occluder with no residual shunt, aortic regurgitation , device [2], which could increase blood transfusion and cause myocardial dysfunction, especially in a pediatric patient. Since Cullen’s report in 1994, percutaneous transcatheter closure of rupture of the RSOV has been developed[3]. However, performed under x-ray guidance, this procedure involves undesired radiation exposure.An acquired Sinus of Valsalva-right ventricle fistula is rare after surgical repair of VSD. Due to the high-pressure gradient across the RSOV to RA, the fistula may cause obvious left-to-right shunt, which will lead to RV overload, or even congestive heart failure. To treat the fistula, repeated open-heart surgery requires a secondary cardiopulmonary bypass[4]. In this procedure, TEE was of utmost importance for (1) sizing the fistula and thus choosing a proper occluder (1–3 mm larger than fistula); (2) understanding the RSOV anatomy with regard to its neighboring structures, namely the aortic valve and tricuspid valve[5]; and (3) most importantly, monitoring AR occurrence and residual shunting. Minimally invasive transthoracic device closure of fistula of RSOV guided by TEE has a number of potential advantages over open surgical repair. First of all, the need for cardiopulmonary bypass can be avoided, which is especially important in cases where there is hemodynamic instability because of fistula of the RSOV. Secondly, a smaller incision can be used compared with the sternotomy of open-heart surgery. Thirdly, transcatheter device closure may be an alternative method but is limited by the radiation exposure in a pediatric patient. It may also potentiate multiple episodes of tachyarrhythmia and subsequent hemodynamic instability during the procedure. Compared with traditional percutaneous closure, TEE guidance entails no radiation damage this technique can cause AR because the occluder lies in the sinus of Valsalva; therefore, in case of any serious consequences such as failure of occlusion, obvious aortic insufficiency, or residual shunt, it is indispensable to perform open-heart surgery immediately to avoid any possible danger. Though this approach allows direct manipulation of the device to prevent interference of the valve apparatus,TEE-guided minimally invasive transthoracic device closure of acquired fistula of RSOV offers a novel alternative to classical surgical repair and percutaneous device closure, since this is a new procedure, appropriate long-term follow-up and management will need to be established.


Circulating tumor cells (CTCs) are cells shed from tumors or metastatic sites and are a potential biomarker for cancer diagnosis, management, and prognostication. The majority of current studies use single or infrequent CTC sampling points. This strategy assumes that changes in CTC number, as well as phenotypic and molecular characteristics, are gradual with time. In reality, little is known today about the actual kinetics of CTC dissemination and phenotypic and molecular changes in the blood of cancer patients. Herein, we show, using clinical case studies and hypothetical simulation models, how sub-optimal CTC sampling may result in misleading observations with clinical consequences, by missing out on significant CTC spikes that occur in between sampling times. Initial studies using highly frequent CTC sampling are necessary to understand the dynamics of CTC dissemination and phenotypic and molecular changes in the blood of cancer patients. Such an improved understanding will enable an optimal, study-specific sampling frequency to be assigned to individual research studies and clinical trials and better inform practical clinical decisions on cancer management strategies for patient benefits. Circulating tumor cells (CTCs), shed from primary or metastatic tumor into the blood , may repCurrently, most studies on CTCs generally employ single or infrequent CTC sampling points –16, withCTCs were first described in 1869 . In thatSubsequent studies enumerating CTCs using different assays, at multiple time points before, during, and after treatment in breast, prostate, and colorectal cancers showed that elevated CTC levels measured at any time during the treatment course predicted for disease progression –15. Convpattern 1—decrease in circulating epithelial tumor cells (CETC) count (>10-fold), pattern 2—marginal changes in count (<10-fold), and pattern 3—an increase or initial decrease with subsequent increase (“saw-toothed” pattern) (>10-fold) [Apart from enhancing prognostication, the use of multiple time points for sampling CTCs may reveal insights into the nature of CTC dissemination in patients’ blood. In 2008, using multiple time point measurements, Pachmann et al. described three typical patterns of changes in CTCs observed for non-metastatic breast cancer patients undergoing adjuvant treatment: 10-fold) . Notably10-fold) . The str10-fold) –9 and suSimilar saw-toothed patterns were reported in subsequent studies employing different CTC isolation strategies, including the clinically validated FDA-approved CellSearch assay –22. TakeThe saw-toothed count pattern suggests that CTC dissemination in the blood is not always continuous but may occur in spurts during therapy. It thus follows that depending on the time of CTC measurement, different CTC counts will be obtained and in turn, different conclusions will be drawn with regard to the nature of the patient’s real-time therapeutic response. This may explain why some studies, employing very few time points of CTC measurement during therapy, appear not to support a correlation between CTC response and tumor response. For example, Pierga et al.  examined(i) Using a typical saw-toothed response curve of a breast cancer patient from response group 3, as presented by Pachmann et al. , we simu(ii) A recent case report of a metastatic breast cancer patient by Marsland and Schuur  demonstr(iii) We monitored our own prostate cancer patient using the CellSearch assay. A spike in CTCs from 1 to 5 was observed within 1 month, which correlated with prostate-specific antigen (PSA) levels and disease progression by PET/CT imaging Fig. . We simu(iv) Another case is our own patient with metastatic prostate cancer, who had weekly CTC sampling at each chemotherapy session. CTCs were isolated using a previously published size-exclusion method . Using tGiven that different CTC count patterns may predict for significantly varied relapse-free survival , it willAll four case studies, using different CTC isolation technologies including the FDA-approved CellSearch assay, demonstrate how patients using a sub-optimal count frequency may end up with different count patterns indicative of different therapeutic responses and varied predictions of relapse-free survival. Depending on the start date and sampling points chosen, there is a good chance that the physician may be misled into concluding that the patient is responding to treatment, when the latter may in fact be developing resistance to therapy. Published studies typically measured CTCs at monthly intervals during therapy to assess the patients’ responses –21. HoweThe above case studies demonstrate the possibility of misleading interpretation of disease progression and treatment responses based on CTC numbers, given insufficient sampling. This assumption is made purely based on CTC total numbers. Apart from the choice of sampling frequency, other factors may further confound attempts to decipher the patients’ actual therapeutic responses based on CTC number. One such factor is the technology used in isolating CTCs from patients’ peripheral blood samples. Currently, the understanding of CTCs is very much dependent on the technological approaches used for their detection and isolation. Many different CTC detection technologies have been developed, including nucleic acid-based detection , 28, detGiven the lack of a gold standard to compare CTC isolation technologies to, it remains a question whether current methods are detecting all CTCs reliably . Even thCTCs may be shed from different sites within the tumor, which are heterogeneous, or from metastases. Molecular characterization of the CTCs such as expression profiling to detect organ-specific metastatic signatures may aid diagnostic and therapeutic strategies . MoleculT790M and other epidermal growth factor receptor (EGFR)-activating mutations during treatment with the tyrosine kinase inhibitor, Gefitinib [T790M missense mutation occurs within the EGFR kinase domain and prevents binding of Gefitinib to this region while preserving catalytic activity [T790M emergence. This, in turn, will enable earlier initiation of subsequent chemotherapy to eradicate the resistant clones, which may improve survival outcome [Advanced CTC analysis, such as genomic profiling, will likely increase the clinical value of CTCs as biomarkers and therapeutic targets. The use of targeted therapies has achieved considerable success; however, many patients relapse due to drug resistance . The detefitinib . The T79activity . In theiactivity . To save outcome , 46. In  outcome –49.Fig. Besides mutation analysis, other molecular assays of CTCs have also been investigated as indicators of treatment response. Using an RNA in situ hybridization assay on CTCs isolated from an index breast cancer patient, Yu et al. demonstrated association of disease progression and treatment failure with EMT . In thisThe case studies presented above highlight the importance of understanding the precise kinetics of CTC dissemination before optimal count frequency can be assigned for large-scale trials and future clinical practice. As shown in the two hypothetical examples above, without appropriate frequency of CTC sampling, molecular analysis of CTCs may lead to potentially wrong assumptions or delayed diagnosis with clinical consequences. To this end, we propose that initial systematic studies involving highly frequent  sampling to understand the kinetics of CTC dissemination in patients of different cancer types, stages of disease, and treatments. A high-resolution “count-scape” will permit visualization of the actual CTC response patterns manifested in different scenarios. In the case of patients with saw-toothed responses, measurement of the range of duration spans of the CTC spikes may then lead to better understanding of the kinetics of CTC spurts and enable determination of the optimal sampling intervals to employ. This systematic approach is perceivably superior to the arbitrary manner in which sampling frequencies are currently being selected and will likely lead to a better gauge of the actual therapeutic responses based on changes in CTC count. While the high cost and resource requirements associated with highly frequent CTC sampling may be prohibitive, these may be overcome in the near future by technological advances leading to low-cost and high-throughput CTC isolation devices. Before CTC-based molecular markers can be translated to the real-world clinical setting, systematic studies with high-frequency sampling of CTCs will likely be required to establish the dynamics of the phenotypic and genetic changes occurring in CTCs.CTCs capture real-time information about the biology of the cancer; changes in CTCs can thus reveal changes in disease status. The ultimate goal in clinical application of CTCs is its incorporation into disease management strategies. CTCs may be used, in conjunction with radiologic imaging, serum tumor markers, and clinical assessment, for real-time monitoring of disease status and therapeutic efficacy. At the moment, the sampling frequency represents a major conundrum and an unresolved unknown in the study and clinical application of CTCs. Current studies in CTC are probably limited in sampling frequency more as a result of cost and resource implications than scientific rationale. To fulfill the full clinical potential of CTCs, an initial study involving high-frequency blood sampling appears to be the unavoidable trip that all CTC researchers must take to ensure there is no loss of crucial information during time-course investigations.Blood was obtained from patient described in case study (2) with approval from the Institutional Review Board (IRB) of National University of Singapore (NUS), following written informed consent.


However, recent studies have shown that the magnitude of mitochondrial genetic effects on the phenotype can vary greatly across environments, with mtDNA polymorphisms commonly entwined in genotype-by-environment (G × E) interactions. Here we test whether the male-sterilizing effects previously associated with the mt:Cyt-b mutation are consistent across three thermal and three nuclear genomic contexts. The effects of this mutation were indeed moderated by the nuclear background and thermal environment, but crucially the fertility of males carrying the mutation was invariably reduced relative to controls. This mutation thus constitutes a promising candidate for the further development of the TFT.Pest species pose major challenges to global economies, ecosystems, and health. Unfortunately, most conventional approaches to pest control remain costly, and temporary in effect. As such, a heritable variant of the Sterile Insect Technique (SIT) was proposed, based on the introduction of mitochondrial DNA mutations into pest populations, which impair male fertility but have no effects on females. Evidence for this “Trojan Female Technique” (TFT) was recently provided, in the form of a mutation in the mitochondrial cytochrome b gene ( Once released, and in the absence of the repressor, expression of the lethal gene is triggered by the expression of a female-specific transcription enhancer, killing females during development while leaving males unscathed. The survival of males enables trans-generational heritability, and feasibility of this repressible approach has been demonstrated in laboratory fruit fly populations6Significant research effort has thus been directed at developing novel SIT variants that promise trans-generational heritability of male sterility, and that ideally require only periodic releases of sterile males to successfully control pest populationsA challenge to SIT variants that remains is to establish effector genes at sufficiently high frequencies within target populations to achieve sustained control. An interesting and alternative approach to SIT, to help the spread of effector genes through target populations, is to cause their biased inheritance through linkage to site-specific selfish genetic elements (“gene drive”)9111114A novel variant of the SIT, which in theory can achieve sustained population suppression over multiple generations in any taxa, and which does not rely on the introduction of novel gene constructs into nature, is the recently proposed Trojan Female Technique (TFT)17Drosophila melanogaster (D. melanogaster) indeed consistently impairs male fertility across a range of diverse nuclear genomic backgroundsmt:Cyt-b), a component of the mitochondrial respiratory complex III25mt:Cyt-b mutation on male fertility in D. melanogaster is well established24mt:Cyt-b mutation for the TFT is particularly interesting given that the same mutation has already been identified in the mt:Cyt-b gene of a number of other species, both vertebrate and invertebrateThe applicability of the TFT to pest control has been theoretically substantiated192122Drosophila, observed patterns were not only shaped by the nuclear background but also by the temperature at which flies were reared2830mt:Cyt-b mutation, assessing whether its fertility outcome for males is modified by interactions with the thermal environment. Because of the importance of the mitonuclear interaction in life-history expression, we further test the thermal interaction across three different non-coevolved nuclear backgrounds.However, a second obstacle that could plausibly stand in the way of further development of the TFT is the scope for genotype-by-environment (GxE) interactions involving sequence polymorphisms in the mtDNA that alter male-fertility phenotypes (cases of “mtDNA-mediated phenotypic plasticity”). Evidence for such plasticity has accumulated in recent years. In early studies that examined mtDNA transmission patterns following experimentally-enforced heteroplasmy in mt:Cyt-b mutation are consistent across varying thermal and nuclear genomic contexts, we placed three different mtDNA haplotypes  alongside three foreign non-coevolved nuclear backgrounds  and measured their effects on male fertility at three different temperatures , have coevolved with the BRO haplotype. This indicates that variably successful mechanisms coded for by the nuclear genome  already exist within the gene pools of tested nuclear backgrounds, and which may be acting to compensate for mtDNA-linked reductions in male fertility. The existence of such modifying mechanisms within the nuclear background is evolutionary plausible, because they would have been selected for over time by the natural occurrence of male fertility-reducing mtDNA variation in the wild populations19mt:Cyt-b gene, in the development of the TFT to aide eradication of pest species.The BRO haplotype was also the mediator of the mitonuclear interactions detected in this study. While male fertility of the two control haplotypes was similar across the three nuclear backgrounds, BRO-linked fertility was much reduced when expressed alongside the DAH nuclear background (reduced by ~72%) relative to CH or LHM  could still theoretically cause marked suppression of natural populations (up to 38%) as its frequency increases , conferring complete male sterility but normal female fertility when expressed alongside the w1118 background27w1118 strain for a further 60 generations to ensure that all four chromosomes in the w1118 nuclear background were isogenic across all mitochondrial strains. The w1118 strain itself is propagated by one pair of full-siblings. Each generation, virgin females of each mitochondrial strain duplicate are backcrossed to males of w1118, thus maintaining nuclear isogenicity across strains. Nuclear isogenicity across mitochondrial strains, coupled with the use of independent duplicates per strain, allowed us to partition true mitochondrial effects from nuclear effects and other sources of variance.The experiment utilised three naturally-occurring mitochondrial haplotypes, sourced from Brownsville USA [BRO], Puerto Montt Chile [PUE], and Zimbabwe [ZIM]1 females that exhibited high genome-wide heterozygosity. All vials were density-controlled throughout the experiment, at 10 pairs of adults and then 80 eggs per vial following ovipositioning, and maintained under constant environmental conditions. All crosses described involved flies of standard-age (3 days), and maintained on a potato-dextrose-agar medium at constant temperature (25.0 ± 0.1 °C), and diurnal cycle (12 h:12 h light:dark).We also created a strain of flies that generated ‘tester’ females of standardized genotype for the male fertility assays. This strain was created by non-reciprocally crossing two near-isogenic lines together, each derived from the same laboratory populationi.e. the grandparents and parents of all flies used in the experiment were reared at the appropriate temperature). The chosen temperature range reflects thermal conditions under which D. melanogaster maintains complete adult fertility1 sons, each of whom possessed one of the three mitochondrial haplotypes , a haploid nuclear copy of the w1118 strain (inherited from the mother), and a paternally-contributed haploid nuclear copy derived from one of the three laboratory populations , were collected as virgins and used as the focal males in the fertility assays  for two generations prior to the assay of male fertility .


Nuclear medicine imaging can provide important complementary information in the management of pediatric patients with neurological diseases. Pre-surgical localization of the epileptogenic focus in medically refractory epilepsy patients is the most common indication for nuclear medicine imaging in pediatric neurology. In patients with temporal lobe epilepsy, nuclear medicine imaging is particularly useful when magnetic resonance imaging findings are normal or its findings are discordant with electroencephalogram findings. In pediatric patients with brain tumors, nuclear medicine imaging can be clinically helpful in the diagnosis, directing biopsy, planning therapy, differentiating tumor recurrence from post-treatment sequelae, and assessment of response to therapy. Among other neurological diseases in which nuclear medicine has proved to be useful are patients with head trauma, inflammatory-infectious diseases and hypoxic-ischemic encephalopathy. Nuclear medicine offers noninvasive imaging methods, namely single-photon emission computed tomography (SPECT) and positron emission tomography (PET) to study functional changes associated with neurological disorders . The mosBrain Single-Photon Emission Computed TomographySPECT is the specific technique used to acquire the three-dimensional distribution of a radiopharmaceutical using a gamma camera. Regional cerebral perfusion can be measured by using technetium-99m (Tc-99m) labeled compounds such as ethyl cysteine dimer (ECD) and hexamethyl propylene amine oxime (HMPAO). These agents cross the blood-brain barrier due to their lipophilic character, and are retained within the brain by their conversion into hydrophilic compounds proportional to regional cerebral blood flow. Their extraction from plasma to brain occurs rapidly within 1-2 minutes and this initial tracer uptake remains almost unchanged for several hours that allow acquisition of a constant image of regional cerebral blood flow at the time of tracer injection. However, there are several differences between these two radiopharmaceuticals in respect to in vitro stability, uptake mechanism, cerebral distribution and dosimetry. When epilepsy is considered, the most important of these factors is the in vitro stability that is 30 min after reconstitution for unstabilized Tc-99m-HMPAO, 4 h for stabilized Tc-99m-HMPAO, and 6 h for Tc-99m-ECD  located Glucose metabolism provides the major energy supply required for brain function, therefore is highly correlated with neuronal activity. Pathophysiologic changes in neuronal activity in disease states cause changes in glucose metabolism that can be detected via the radiolabelled glucose analogue fluorine-18 fluorodeoxyglucose (F-18 FDG), which is currently the most commonly used tracer for brain PET imaging. The most common indications for brain F-18 FDG PET imaging include preoperative evaluation of partial epilepsy patients to identify the functional deficit zone, early diagnosis and differential diagnosis of dementing disorders, differential diagnosis of cerebral tumors, and detection of viable tumor tissue following therapy . HoweverSince brain perfusion and metabolism are coupled with neuronal function, radiotracer injection should be done in a controlled environment to keep neuronal activation at a minimum. Patients should be positioned comfortably in a quiet, dimly lit room, with an intravenous line inserted at least 10 min before administration of the radiotracer. Patients should be instructed not to speak, read or be active otherwise at the time of injection and during the uptake phase, which is at least 20 min for F-18 FDG  can benefit from nuclear medicine imaging ,31,32,33In pediatric TLE, the most common underlying pathologies are mesial temporal sclerosis, focal cortical dysplasia and developmental tumors . The usuThe localization of the epileptogenic focus in patients with intractable extratemporal lobe epilepsy (ETLE) generally requires more extensive noninvasive and invasive EEG monitoring. The main role of functional imaging with radioisotopes in lesional or non-lesional ETLE is to guide the surgeon in placing the subdural electrodes for invasive EEG monitoring. However, the sensitivity of interictal F-18 FDG PET in detecting the epileptogenic focus in ETLE is not as high as in patients with TLE ,44. For Brain tumors are the most common solid tumors in childhood. The management of a child with a brain tumor depends on both the histological features of the tumor and its location within the nervous system. Although surgery is the mainstay of therapy in primary brain tumors, centrally localized tumors can rarely be totally resected and additional chemoradiotherapy is usually required. MRI is generally used to monitor the effects of treatment on the tumor and to detect recurrence. However, MRI is impaired by changes in brain tissue related to surgery and chemoradiotherapy that may cause false positive contrast enhancement. PET and SPECT imagings are suggested for monitoring treatment effects and detecting recurrence . When quF-18 FDG is the most commonly used PET tracer for metabolic studies of brain tumors. It can be used as a diagnostic tool since malignant tumors have a higher F-18 FDG uptake and benign tumors have a lower F-18 FDG uptake than normal brain tissue. Additionally, F-18 FDG PET can be used as a tool to improve the quality of brain tumor biopsies by metabolically showing the most active part of the tumor . AlthougPediatric brain injury is among the most common causes of death or permanent disability in children. Brain injury can result in various neuropathological changes that affect cerebral blood flow and metabolism; that is why functional neuroimaging may show changes that occur in response to neurologic damage even in regions that appear structurally normal on CT or MRI and provide a more accurate diagnosis of neurologic damage and clinical outcome, especially when there is no clear-cut relationship between the anatomic and clinical findings ,63,64,65Rasmussen’s encephalitis, which is a childhood disease of unknown etiology and presenting with resistant epilepsy, progressive hemiparesis and cognitive disorders, is among the most commonly investigated inflammatory neurological diseases in children by SPECT and PET. Typically only one hemisphere is affected in Rasmussen’s encephalitis and cortical atrophy develops in the affected hemisphere . Brain SIn newborn patients with hypoxic-ischemic encephalopathy, brain perfusion SPECT and F-18 FDG PET scan findings have been compared with neuro-developmental outcomes ,71,72. SNuclear medicine methods can contribute to the management of pediatric patients with neurological diseases. Previous studies have demonstrated that nuclear medicine imaging provided complementary information in pre-surgical localization of the epileptogenic focus in medically refractory epilepsy patients as well as in diagnosis and surveillance of patients with brain tumors. Other indications of nuclear medicine in pediatric neurology are the evaluation of patients with head trauma, inflammatory-infectious central nervous system diseases, and hypoxic-ischemic encephalopathy. Although brain perfusion and metabolism are the most commonly studied functions, several other more specific tracers exist for assessment of various neuronal functions.


The spatial distribution of forage resources is a major driver of animal movement patterns. Understanding where animals forage is important for the conservation of multi-species communities, since interspecific competition can emerge if different species use the same depletable resources. However, determining forage resources in a spatially continuous fashion in alpine grasslands at high spatial resolution was challenging up to now, because terrain heterogeneity causes vegetation characteristics to vary at small spatial scales, and methods for detection of behavioural phases in animal movement patterns were not widely available. We delineated areas coupled to the foraging behaviour of three sympatric ungulate species  using Time Local Convex Hull (T-LoCoH), a non-parametric utilisation distribution method incorporating spatial and temporal autocorrelation structure of GPS data. We used resource maps of plant biomass and plant nitrogen content derived from high-resolution airborne imaging spectroscopy data, and multinomial logistic regression to compare the foraging areas of the three ungulate species.We found significant differences in plant biomass and plant nitrogen content between the core foraging areas of chamois, ibex and red deer. Core foraging areas of chamois were characterised by low plant biomass and low to medium plant nitrogen content. Core foraging areas of ibex were, in contrast, characterised by high plant nitrogen content, but varied in plant biomass, and core foraging areas of red deer had high plant biomass, but varied in plant nitrogen content.Previous studies carried out in the same study area found no difference in forage consumed by chamois, ibex and red deer. Methodologically, those studies were based on micro-histological analysis of plant fragments identifying them to plant family or functional type level. However, vegetation properties such as productivity (biomass) or plant nutrient content can vary within vegetation communities, especially in highly heterogeneous landscapes. Thus, the combination of high spatial resolution resource maps with a utilisation distribution method allowing to generate behavioural maps (T-LoCoH) provides new insights into the foraging ecology of the three sympatric species, important for their conservation and to monitor expected future changes.The online version of this article (doi:10.1186/s40462-015-0033-x) contains supplementary material, which is available to authorized users. How ecologically similar species coexist in a shared habitat is a fundamental question in ecology . ResourcRupicapra rupicapra L.), ibex (Capra ibex L.) and red deer (Cervus elaphus L.) as mixed feeders, with chamois being closer to browsers, ibex closer to grazers and red deer in between  ). If the animal was captured or recaptured during the 43 day time window around the APEX flights, the first and last days of data collection were excluded from analysis. GPS data were screened for unrealistic movement following the method of Bjørneraas et al. , with liWe used the T-LoCoH package in R , a non-pThe number of nearest neighbours can be defined by selecting the “k” closest points in space and time (“k-method”), the points within a defined time-scaled radius “r” (“r-method”) or by identifying the nearest neighbours up to a cumulative distance “a” in space and time (“a-method”). We decided to use the “a-method” as this method is better suited for studies where both, high and low point densities of GPS locations can be expected . As befoWhile there are guidelines available for selecting the weight placed on the time-component  and the threshold for nearest neighbour selection  , the parWe fitted multinomial logistic regression models using the three ungulate species as the response and plant biomass and plant N content in the animals’ core foraging areas as predictor variables. We rescaled plant biomass to a level similar to plant N content by dividing all biomass values by 100 (BiomRS = Biomass/100). As candidate models we chose i) the two models containing only one predictor variable (plant biomass or plant N content), ii) the main effects model containing both terms (plant biomass and plant N content), iii) the model including both terms plus their interaction and iv) the intercept-only model. We selected the best model based on differences of AIC (Δ AIC) and confirmed our selection using the likelihood ratio test. To evaluate model fit we calculated the Hosmer-Lemeshow goodness-of-fit statistic and the area under curve (AUC) of the receiver-operating characteristic (ROC) for each of the two logits separately . The ROCThe SRI models of grassland vegetation developed from APEX IS data predicted plant biomass and plant N content with high to moderately high predictive power Table . General−2 fresh weight, Figures The core foraging areas of chamois were characterised by generally low plant biomass (< 200 g.mCapreolus capreolus L.) in Southern Germany [Ovis ammon musimon Pallas) in the Western Alps [Previous studies conducted in the Trupchun valley found no difference in spring and summer forage composition between chamois, ibex and red deer when using micro-histological analysis of plant fragments in faecal pellets -24. Like Germany , with moern Alps  and withern Alps . While oern Alps , it coulern Alps . Similarern Alps , but couern Alps . This maern Alps ,93,94, aThe studies mentioned above used micro-histological analyses of faecal pellets and thus identified forage remains at the level of plant functional groups or plant families. However, the ratios of the two main forage components, graminoids and forbs, varied considerably within the ungulate species studied, which suggests that the animals have some flexibility in their dietary choices. Moreover, ungulates are assumed to partition forage resources at levels below the scale of vegetation types , and sevFinding core foraging areas of chamois predominantly where plant biomass was low, red deer core foraging areas where plant biomass was high and ibex in between is in line with traditional feeding type definitions . RegardiIn contrast to chamois, vegetation in the core foraging areas of ibex was characterised by high plant N content and variable plant biomass. Generally, we expected ibex to forage in rocky terrain with little, but nutrient-rich vegetation. While our results suggested that ibex did indeed forage in areas where plant biomass was low but of high nutritious value (high plant N content), we also found ibex core foraging areas in the highest quality meadows of the Trupchun valley where both plant biomass and plant N content were high. Generally, terrain roughness and slope create a template of risk ,97, in wThe core foraging areas of red deer were always located on the rather flat meadows where the animals are expected to be able to cover their forage intake needs as plant biomass is high. Red deer inhabit predominantly open, flat terrain, have good running skills and thus withdraw themselves from predator attacks by using areas with high lateral cover, such as areas with forest or tall-growing shrubs. Similar to ibex, red deer might have abandoned part of their anti-predator behaviour as they can be observed grazing, resting and ruminating on the alpine meadows of the Trupchun valley during daytime ,68. HoweDue to the fixed dates of the APEX IS data acquisition, the results of our study represent the animals’ behaviour during a specific time, i.e. during early summer. However, forage quantity and quality are expected to influence ungulate movement patterns in our study area in particular during this time of the year (peak of the plant growing season), when females have to nourish their offspring, and all individuals have to build up winter reserves. Naturally, the quantity and quality of forage resources will change during the course of the year and therefore also the ungulates’ habitat use patterns can be expected to change ,104. TheThe distribution of vegetation quantity and quality influences the space use of herbivores on several spatial and temporal scales ,107. LarWe detected significant differences in plant biomass and plant N content in the core foraging areas of sympatric chamois, ibex and red deer when combining resource maps developed from airborne imaging spectroscopy data with behavioural maps developed using the T-LoCoH algorithm: T-LoCoH enables to detect behavioural phases in GPS data by making use of their temporal and spatial autocorrelation. The combination of behavioural and resource maps proved to be valuable for studying a classical issue in resource ecology, resource partitioning between sympatric species. For the future, we expect that the development of remote sensing instruments with increased spatial resolution and temporal flexibility together with highly accurate and short interval GPS systems will continue to deepen our understanding of the foraging ecology of multi-species communities.


The purpose of this essay is to increase the awareness on what patients, families andthose involved in the treatment and follow-up can face in the long run regarding thesurvivors of the Fontan operation (FO) and to provide some clues to diminish thedeleterious effects of the single ventricle (SV) physiology. Although a significantnumber of patients survive and are initially asymptomatic, most probably due toadaptation to their limitations, an active approach without being pessimistic is neededin order to prevent problems and thereby improve the long-term prognosis of thesepatients.Those who believe thatone ventricle can drive blood full of spirit into the body and the lungs likewise,are heretics. They forget that nature, being divine, never gave a heart to any wherethere was no need." When Fontan and Baudet published their procedure theyadvised us that "this procedure is not an anatomical correction, which wouldrequire the creation of a right ventricle, but a way of physiological pulmonaryblood flow restoration, with suppression of right and left blood flowmixing".1 They alsodescribed the "Ten commandments", a list of precise recommendations regarding theindications for surgery.2 Some of theproblems that we see today are the result of not following their guidelines. Althoughthey clearly defined the main purpose of this procedure, they could not anticipate thelong-term implications of this improvement.When William Harvey described the circulation he stated: "elevated central venous pressure, and 2)suboptimal cardiac output, relative to the normal two-ventriclestate due to: a) impaired chronotropic response to exercise; b) decreased capability totransport a normal blood volume across the pulmonary vascular bed, resulting in reducedventricular filling and low stroke volume; c) inability to adequately increase strokevolume during periods of increased demand; and, d) elevated systemic vascular resistancewith deprived tissue perfusion and anaerobic metabolism.More than forty years later, we are witnessing the consequences of not having asub-pulmonary ventricle. Although clinical experience shows that to survive we do notneed a pumping chamber to drive blood into the lungs, the non-pulsatile flow of bothvena cava directly connected to the pulmonary circulation results in the followingunintended consequences: 1) 8 However, despite the remarkable improvements inquality of life and prognosis of patients treated by the FO, there is a decreasedexercise capacity and a suboptimal ventricular performance, subsequent to thereduced preload of the functional SV. In addition, these patients often developscoliosis, kyphosis, have small lungs and consequently a restrictive pulmonarypattern due to previous thoracic surgical procedures. Altogether, it affectsdifferent subsystems with a negative impact on functional status, quality of lifeand the long-term transplant-free survival ratio. Multiple studies assessing theresults of the FO demonstrated a decrease in survival with a continuous attrition 15years after the procedure, regardless of the surgical type of cavopulmonaryconnection.9 In a recentsingle-institutional study, the actuarial freedom from death or transplantation was87%, 83%, and 70% at 15, 20, and 25 years respectively after surgery. In this group,death was sudden and unexplained in 9%, thromboembolic in 8%, and heartfailure-related in 7%.10 Inanother study assessing morphologically left SV, actuarial survival was 73% at 15years. Atrial arrhythmias were present in 57%, protein-losing enteropathy in 9%, andthromboembolic events in 6%. In other words, odds are 1 out of 4 that a child afterFO will be dead by the time he or she reaches their late 20s.11 In a large multi-center cohort,the Pediatric Heart Network (PHN) reviewed 546 children that were on average 11.9years of age at the time of the study and 8.5 years after the FO. Stroke orthromboembolism was seen in 8% of the patients, exercise performance was abnormaland peak oxygen consumption was only 65% of the predicted for age and gender in arelatively young group.12Adolescents ("older" patients) fared worse than the younger children, suggesting atime-related decrement in functionality.13 In another PHN study, parent-reported patient morbiditiesincluded deficits in vision in 33%, speech in 27%, and hearing in 7%, as well asproblems with attention in 46%, learning in 43%, development in 24%, behavior in23%, anxiety in 17%, and depression in 8%.14Presently, this operation is the gold standard destination for patients with SVphysiology. Along the years, techniques and strategies have evolved from theoriginal right atrium to pulmonary artery connection to the lateral tunnel and theextra-cardiac conduit, including staged procedures and fenestrations. Better resultswith this operation have been assigned to these technical modifications. There aremany data showing excellent early and long-term outcome with good survivalrate.15recently reported suboptimal cardiac function, diminished lung volumes andcapacities, as well as reduced respiratory muscle strength in asymptomatic patients- the so-called "perfect Fontan".16 These areimportant components of a complex system in which performance and outcomes depend onintricate dynamic interactions that could explain the deficiencies found in the latepostoperative period. In other words, despite the low early mortality, when weassess long-term morbidities, the number of patients that are free from problems islow. These findings cannot be ignored, and certainly do not reflect a successfulmanagement strategy for patients with single-ventricle physiology.In our center, Turquetto et al.In summary, despite the good early results, long-term survivors could experience someof the following complications:ArrhythmiasThromboembolismDelayed somatic growthDeferred pubertal developmentProtein losing enteropathy (PLE)Plastic bronchitis (PB)Exercise intoleranceLiver fibrosisRenal dysfunctionVenous insufficiency+2. Urine calcium/creatinine for nutritional assessment,B-natriuretic peptide, cystatin C and basic immune panel should also beperformed.17The above mentioned issues justify a regular and careful follow-up of these patientsevery three to four-year intervals, with major testing at 10 years after FO. Thehealth status of children and adolescents after FO is suboptimal and the managementof the late complications represents a significant challenge. If possible, everypatient should have serial echocardiogram studies, cardiopulmonary test, abdominalultrasound, whole body dual energy X-ray absorbtiometry scan and complete bloodanalysis including CBC with differential, electrolytes, liver enzymes, GGT, totalprotein, albumin, parathyroid hormone, vitamin D 25-hydroxy and serum ionizedCa18In the late follow-up, we might face a failing Fontan. Failing Fontan patients havetwo modes of presentation: a) impaired ventricular function and b) those withpreserved ventricular function, but with failing subsystems such as PLE and PB. Asmany as two thirds of adult Fontan patients who die or require transplantation do sowith preserved ventricular function.19The insufficient availability of donors and the associated morbidities withimmunosuppression make it imperative that this scarce resource is used appropriatelyby optimizing the timing of transplantation. Therefore, it is important to identifyhigh-risk patients with failing Fontan physiology and diastolic dysfunction thatmight benefit from other modes of therapy. Ventricular assist devices (VAD) that maybridge patients more successfully are needed to better prepare the SV circulationover months in order to improve transplant outcomes through better patientselection.20Due to the increasing amount of patients that are being palliated with FO, the numberof children, adolescents, and young adults requiring late rescue therapy with hearttransplantation will increase.21Children and adults with previous procedures undergoing heart transplantation requiremore complicated operations that should be performed by skilled surgeons. Outcomesof heart transplantation in children with Congenital Heart Disease (CHD) haverepeatedly been shown to be inferior to those in children withcardiomyopathy.22 Several factors have contributedto the substandard results in the Fontan group, including allosensitization,pulmonary hypertension, challenging operation because of multiple priorsternotomies, complex venous anatomy, requirement of concomitant pulmonary arteryreconstruction and the presence of collaterals with subsequent risk of bleeding. Inaddition, poor clinical condition due to protein-losing enteropathy, malnutrition,liver and kidney dysfunction are aggravated conditions.22 We speculate that early referral in betterclinical conditions could be responsible for the outstanding results described inthis recent publication.Although several centers have described that heart transplantation after the FO isassociated with poorer outcomes when compared with those due to other forms of CHD,recent report has shown outstanding outcomes in the former group, comparable tothose in children receiving transplants for cardiomyopathy.Considering the late results of SV palliation, we should strongly consider followingthe right indications and timing for each step, not based on symptoms, butprogrammed in advance. Following the internationally recognized algorithm, early inlife we ought to band the pulmonary artery to protect the pulmonary vasculature andprevent high pulmonary vascular resistance in those with high pulmonary blood flow,or to create a shunt in those with hypoxemia. The next step should be to diminishthe SV volume overload by connecting the superior vena cava to the pulmonary artery- bidirectional Glenn operation - at 3 to 6 months and completing the FO at 2 to 4years of age.At the end, we might face two different undesirable scenarios:Pump failure that will need heart transplantation at the right time andbefore clinical deterioration.23 The latter has a greater risk of death whencompared with the group that presents with poor ventricularfunction.Those with the negative impact of the SV physiology on other subsystems,due to the lack of sub-pulmonary ventricle, which can benefit from a VADto gain time to optimize patient's condition fortransplantation.Further studies need to be performed to better understand the "unnatural" state ofFontan patients. Strategies targeted toward improving cardiac output and reducingcentral venous pressure can improve their overall well-being and mitigate thedeleterious impact of the new physiology. Further research on methods to improve thecirculation through additional interventions - pharmacological, mechanical and evenexercise training - is required.