There is a growing body of literature supporting positive associations between natural environments and better health. The type, quality and quantity of green and blue space (‘green-space’) in proximity to the home might be particularly important for less mobile populations, such as for some older people. However, considerations of measurement and definition of green-space, beyond single aggregated metrics, are rare. This constitutes a major source of uncertainty in current understanding of public health benefits derived from natural environments. We aimed to improve our understanding of how such benefits are conferred to different demographic groups through a comprehensive evaluation of the physical and spatial characteristics of urban green infrastructure.We employed a green infrastructure (GI) approach combining a high-resolution spatial dataset of land-cover and function with area-level demographic and socio-economic data. This allowed for a comprehensive characterization of a densely populated, polycentric city-region. We produced multiple GI attributes including, for example, urban vegetation health. We used a series of step-wise multi-level regression analyses to test associations between population chronic morbidity and the functional, physical and spatial components of GI across an urban socio-demographic gradient.GI attributes demonstrated associations with health in all socio-demographic contexts even where associations between health and overall green cover were non-significant. Associations varied by urban socio-demographic group. For areas characterised by having higher proportions of older people (‘older neighbourhoods’), associations with better health were exhibited by land-cover diversity, informal greenery and patch size in high income areas and by proximity to public parks and recreation land in low income areas. Quality of GI was a significant predictor of good health in areas of low income and low GI cover. Proximity of publicly accessible GI was also significant.The influence of urban GI on population health is mediated by green-space form, quantity, accessibility, and vegetation health. People in urban neighbourhoods that are characterised by lower income and older age populations are disproportionately healthy if their neighbourhoods contain accessible, good quality public green-space. This has implications for strategies to decrease health inequalities and inform international initiatives, such as the World Health Organisation’s Age-Friendly Cities programme. Exposure to the natural environment has long been recognised as an important component of human well-being. The continued expansion of urban areas and sustained rural-to-urban migration have stimulated recent research on the role that green-space might play in urban planning and policy in a public health context . In lineA recent review by Zhang et al.  suggestsWe argue that operationalizing a GI approach through the use of spatially and thematically detailed data sets, encompassing the range of urban landscape characteristics pertinent to health and wellbeing, is needed to advance the field towards more robust implementations of nature-based public health interventions. To this end, our study employs a suite of social and physical environmental variables in order to build a comprehensive picture of links between characteristics of urban green infrastructure and local health status to fill an important science-policy gap. We predicted that GI should exhibit varying associations with health as a function of land-use and land-cover attributes, and socio-demographic context. For example, the presence of local accessible GI may provide particular benefit to the least physically mobile populations. Given that physical mobility can be constrained in both older and more economically deprived groups, members of the population falling into both of these categories should logically rely most strongly on nearby green infrastructure for nature experience. Moreover, such sectors of the population also tend to be the most vulnerable to climate-related hazards for which the presence of GI is a mitigating factor . Related2 with a population of 2.8 million as of 2016 [Spatial data were obtained from a novel landscape dataset representing urban green infrastructure (Integrated Landscape Map: ILM) . These d of 2016 . The dat of 2016 , digitiz of 2016  and land of 2016 –32. Figu1 ranged from 0.62 % to 98.81 % with a mean of 57.45% (SD 19.54). LSOAs are census reporting units for small area statistics in England and Wales with a mean population of 1666 persons for which socio-economic data were also obtained [Green land-use in the ILM dataset includes public parks and recreation , amenity land , private gardens, institutional land, previously-developed land, peri-urban and informal urban greenery . Land-cover is characterised as built, ground layer vegetation (grass lawns and other ground flora), field layer vegetation (forbs and shrubs), tree canopy, and water. Percentage cover by individual land-uses and land-covers were calculated for each of the 1673 Lower Super Output Areas (LSOAs) neighbourhoods within Greater Manchester in ArcGIS 10.4.1 . Total gobtained .Built-cover within land-uses otherwise considered as green infrastructure has been highlighted as a considerable problem for the ability of GI to provide ecosystem benefits. Recently, work by Baker et al. exposed the significant degree of built cover found in UK domestic gardens (on average 50%) . Elsewhe2 of patches of non-built land-cover) and the metric effective mesh size (Meff) provides a measure of the total area of connected GI in a given area. In addition, patch size of individual land-uses was determined through OS MasterMap data [2).A range of metrics underpinning GI performance  values. This vegetation-specific NDVI (vNDVI) was used based on the assumption that land-cover with a higher level of vigour indicates healthy and well maintained vegetation. Whereas mean NDVI values, calculated for example at the neighbourhood level, are commonly employed elsewhere in epidemiological studies as a measure of greenness, the metric, used in this way, is more closely associated with vegetation density and therefore a metric of relative abundance. However by isolating pixels classified as vegetation in our dataset, and using this layer as a mask in our NVDI calculation, our aim was to delineate the greenness of individual patches of vegetation as a measure of GI health. At the LSOA level, mean vNDVI was only weakly correlated with percentage green space cover (r2 = 0.16), overall NDVI (r2 = 0.20) and even more weakly with cover by individual vegetation types (r2 0.03 – 0.09). This confirmed that our metric was not acting as a surrogate for vegetation extent (i.e. quantity) but as a reliable measure of the vigour of existing vegetation . This approach has been used elsewhere to measure vegetation health and has proven to be effective in discriminating between levels of vegetation quality across urbanisation and socio-economic gradients [A measure of vegetation health (or vigour), was also obtained by isolating pixels classed as green-cover in the ILM and calculating the mean Normalized Difference Vegetation Index LSOAˉradients .A measure of the percentage population per LSOA living in close proximity to public parks and recreation land was calculated using high resolution (10 m) population data from the University of Southampton’s OpenPopGrid  dataset Comparative Illness and Disability Ratio (CIDR) of the Health and Disability Deprivation domain within the IMD dataset provides a measure of chronic morbidity at the neighbourhood level. This indicator is derived from the proportion of the local population suffering from chronic ill-health and is principally calculated from data on income benefits received as a consequence of long-term illness. The index is age- and sex-standardized thereby allowing us to make realistic comparisons between different demographic groups. The index is derived from a range of work and pension-related income benefits. Individuals can only claim one such benefit at a time and working-age and retirement-age claims are included [r2 = 0.91). LSOAs in Greater Manchester had a mean CIDR score of 144 (SD 45) compared to 112 (SD 41) for all 32,844 LSOAs in England highlighting the relatively high levels of health deprivation in the city-region. Greater Manchester therefore provides a suitable study area to investigate the potential mitigation of population morbidity through the presence of green infrastructure. Data on neighbourhood (LSOA) population age ranges were downloaded from the UK Data Service for the UK 2011 census [Socio-economic and health data were obtained from the English Indices of Multiple Deprivation (IMD) 2015 . This daincluded . Therefoincluded ). This dincluded . Of the 1 census .2 statistic). In step three, to test for the influence of land-cover and patch size associated with individual land-use types, mean percentage non-built cover and patch size  of gardens, institutional land, amenity and public parks and recreation classes  per LSOA were then entered into a final series of models as additional independent variables to those in step two. All models were adjusted for income, employment, crime, education, and barriers to housing and services deprivation. Moran’s I statistics were computed for step one models to test for the influence of spatial auto-correlation in model residuals which can violate assumptions of variable independence in ordinary least squares regression models. A summary description of predictor variables and their inclusion in the analysis is provided in Table Associations between GI variables and chronic morbidity were explored through multi-level linear regression analyses in a three-step process. In step one overall non-built land-cover was regressed on chronic morbidity stratifying by tercile groups for income  and proportion of the adult population over sixty years of age, hereafter: younger neighbourhoods (0 – 17.3% (bottom tercile) adults ≥ 60 years), mid-age range neighbourhoods (17.4 – 23.6 % adults ≥ 60 years) and older neighbourhoods (> 23.6 % (top tercile) adults ≥ 60 years). In step two, a second series of models were developed where individual, disaggregated GI attributes  and proximity variables) were added in a forward-stepwise fashion until a final best-fit model was established  of which 41·7% (SD 15·3) was non-built cover) compared to older and higher income areas (mean size 227 m2 (SD 308) of which 58·3% (SD 14·3) was non-built cover, Table Overall GI quantity (green cover) increased with both income and age, with the lowest mean cover in low income, younger neighbourhoods  and highest mean cover in high income older neighbourhoods . Provision of public parks and recreation land and percentage of the population in close proximity to this land-use was generally greatest in middle income areas whereas amenity land was most abundant in lower income areas. Private gardens were greatest, in terms of extent, size and proportion of non-built cover, in higher income older neighbourhoods. Low income younger neighbourhoods exhibited the smallest mean garden size (77 mTable I < 0.3) or non-significant levels of non-stationarity in regression models, suggesting the analysis was robust to spatial effects and comparable to those found elsewhere [The strongest associations between percentage non-built cover and health were seen in younger neighbourhoods of medium or high income. Analyses for these groups also produced the lowest model fit with highest model r-squared values exhibited by analysis of older age-group. Similarly, spatial-autocorrelation  exhibited variability across socio-demographic groups. Higher income groups were generally subject to a higher influence by spatial auto-correlation, particularly in mid-age range and older neighbourhoods. All other socio-demographic groups however exhibited low . However, in stratified models, garden size was only significant in high income older neighbourhoods . The proportion of non-built land-cover in gardens was relevant in three cases: in high income mid-age range neighbourhoods  and in middle  and high  income older neighbourhoods. Likewise, the proportion of non-built cover in amenity spaces was prominent in the all-population model  but subsequently found to be significant only in middle income older neighbourhoods . Model fits were generally higher when measures of size and the proportion of non-built cover of these land-uses were considered  model , the latter also positively correlating with health for this group. It follows that in the most built up areas, large green spaces may act as potential buffers against chronic ill-health by providing opportunities for recreation  and reliPercentage cover by amenity land-use demonstrated non-significant associations with health. This was perhaps surprising given that this land-use was generally most abundant in low income areas and that health outcomes in such areas in particular have been shown to be sensitive to GI cover , 49. ThiGarden cover exhibited a positive relationship with health in high income, middle age-range populations but a negative association in the low income younger neighbourhoods. These two groups showed great disparity in terms of percentage cover by domestic gardens, with the latter exhibiting the highest mean cover for the study Table . SimilarThis study had some limitations. Firstly, as a correlational study, we cannot rule out the effects of self-selection of healthier people into greener areas. However, the comprehensive range of GI variables used to quantify the natural environment and the stratified approach based on socio-demographic groups used here means that the effects of self-selection on the results are less likely. Secondly, the presence of ecological fallacy cannot be ruled out and further analysis at the individual level would be helpful to confirm the validity of our results. For example, we did not include any consideration of individual user preferences such how older people choose to use their leisure time from the point of view of nature engagement. Thirdly, it was not clear whether unique associations between environmental variables and socio-demographic characteristics occurred as a function of social or physical environmental processes. For example, ground vegetation, tree cover and SHDI were uniquely associated with better health in high income older neighbourhoods. Our approach cannot confirm, however, if this resulted from a particular affinity between this socio-demographic group and these characteristics or simply from particularly high mean values for these variables in such neighbourhoods. However, that other areas with similar mean values did not exhibit similar results suggests that socio-demographic factors may play at least a partial role in such associations.Our GI dataset did not include other small-scale well established examples of urban green infrastructure such as bio-swales and green roofs that also provide important regulating functions (i.e. flood retention or reduced energy demand). Such technical ‘nature-based solutions’  are inclWe took a GI approach to the analysis of associations between health  and the natural environment in urban areas. Our study considered the widest range of GI attributes yet examined, namely land-cover, land-use, landscape attributes  and proximity variables. All of our proposed hypotheses were supported to some degree by the results of this study. Firstly, older people in low income urban areas appear to be disproportionately healthier if those areas are served by local accessible green spaces. Secondly, the quality  as well as quantity (percentage cover by vegetation) associated with urban green infrastructure predicted health outcomes. Attributes such as size, non-built cover and land-cover diversity likewise appeared to be relevant to our analysis of patterns in neighbourhood-scale chronic morbidity. In addition, our over-arching supposition that associations between health and GI are moderated by socio-demographic contexts was upheld. An emphasis on publicly accessible GI, maintenance of healthy vegetation and diverse, nearby green spaces represent promising opportunities for public health interventions particularly in areas characterised by low income and high proportions of older people. These findings have direct consequences for urban planning and may help to inform international initiatives, such as the World Health Organisation’s Age-Friendly Cities programme.Additional file 1.


An ideal sample source is critical for more reliable and sensitive early detection of nucleic acid changes associated with breast cancer. Breast milk (BM) is a good noninvasive origin for genetic testing of early breast cancer, but cells in BM are easily disintegrated. So we investigate here whether cell-free nucleic acid (cfNA) exists in BM in a more stable form and whether the quality of BM cfNA is good enough for genetic testing. z-test based on whole genome sequencing data were used to determine the integrity of genetic information in BM cfNA.  A self-designed qRT-PCR method was used to measure the existence and abundance of cfDNA. Quality of cfDNA and cfRNA were detected by capillary electrophoresis. Whole genome bisulfite sequencing and miRNA sequencing were used to explore the sources of cfDNA and cell-free miRNA in BM. The copy number analysis and  We found that cell-free DNA and miRNA exist in the studied breast milk samples in a stable form that can tolerate incubation of BM at room temperature for at least 7 days. These cell-free nucleic acids come mainly from breast-derived cells and contain genetic information as good integrity as in BM cells. We further listed some candidate miRNAs as potential biomarkers for research of early breast cancer screening by analysis of previous reports and our data.  Our results suggest that cfDNA and cell-free miRNA in BM might be new noninvasive sample sources for finding early alterations of nucleic acid associated with the initiation and progression of breast cancer. Breast cancer is one of the commonly diagnosed diseases and is the leading cause of all cancer death in women worldwide. Its incidence under 50 years old is on the rise, particularly in developing countries , 2. In CScreening and early diagnosis for breast cancer is considered effective for reducing breast cancer-related mortalities. Yet mammography, the traditional screening method, is less sensitive on very early breast cancer and not recommended as a routine method for women younger than 50 years old [During the past decade, increasing attention has been paid to detecting cell-free nucleic acid (cfNA) in plasma, including cell-free DNA (cfDNA) and microRNA (miRNA), which are considered useful for early detection of cancer , 7. HoweBreast milk (BM) is a natural noninvasive sample source that has a potential in the study of breast cancer risk. Lactocytes and epithelial cells in fresh BM may offer a noninvasive origin of nucleic acid for examining genetic, epigenetic, or gene expression changes associated with breast cancer , 12. ButIn this study, we found that abundant and high-quality cfDNA and cell-free miRNA exist in BM in a relatively stable form at RT. Our results show that cfNAs in BM mainly come from breast-derived cells, including lactocytes (milk secretory cells) and myoepithelial cells , and contain genetic information similar to that in BM cells. Perhaps, these characteristics might make BM cfDNA and cell-free miRNA new noninvasive sample sources for screening early alterations of nucleic acid associated with the initiation and progression of breast cancer.Human breast milk samples from healthy donors were obtained with informed consent under institutional review board-approved protocols. Samples were derived from local healthy donors from Beijing. Details of sample collection and processing are provided in Supplemental Text  method, and extraction of genomic DNA (gDNA) was via the QIAamp DNA mini kit (Qiagen). BM cfDNA was extracted using a cfDNA purification kit (SummerBio). BM cfRNA isolation was carried out using a cfRNA purification kit (SummerBio). All nucleic acid extraction processes were carried out following the manufacturer's procedure.BM cfDNA abundance was measured by using a self-designed qRT-PCR method. Full details of the experiments and result processing are provided in Supplemental Text.cfDNA and cfRNA quality was detected by capillary electrophoresis using Qsep100 Bio-Fragment Analyzer (BiOptic) and Agilent 2100 Bioanalyzer, respectively.RNA samples from different donors were subjected to RT-qPCR assays to determine the difference of expression levels of LALBA, K-CSN, B-CSN, ELF5, and TACSTD2, and PPBP, CD99, ITGB3, RGS18, and CD45. Forward and reverse primers for qPCR are listed in Supplemental Table miRNA sequencing and whole genome bisulfite sequencing (WGBS) were performed by LC Sciences . Details of library generation and NGS data analysis are provided in Supplemental Text. The whole genome sequencing (WGS) library of cfDNA was generated by using a NEB Next Ultra DNA Library Prep Kit for Illumina (NEB) according to the manufacturer's recommended protocol. Processes of quality control, sequencing, and data analysis are provided in Supplemental Text in detail.Statistical information is indicated in the text or figure legends.Fresh breast milk (BM) contains cells that may offer a noninvasive source for genetic testing , 13. ButPrevious reports reveal that the population of BM cells was composed of breast-derived cells and blood-derived cells, and breast-derived cells, including lactocytes (milk secretory cells) and myoepithelial cells , make up the majority of the population of BM cells , 15. In To investigate whether BM cfDNA is mainly derived from breast-derived cells, whole genome bisulfite sequencing (WGBS) was carried out to compare epigenetic similarity between gDNA of BM cells (from donor 6) and BM cfDNA (from donors 6 and 8). The slide window method was applied for methylation level analysis. With window size = 1000 bp and step size = 500 bp, the sum of methylated and unmethylated C read counts were calculated in each window. The methylation level (ML) for C sites in each window is defined as ML = mC reads/(mC reads + C reads). To eliminate the unreliability of data with low sequencing depth, slide windows with total C read counts less than 3000 were filtered. Results show that the methylation levels in different regions of each chromosome are highly consistent between samples Figures . Pearsonz-test is a method used to analyze the aneuploid based on WGS data. By this method, a z-score is calculated to measure deviation of a chromosomal dosage from a set of euploid control samples [z-test was utilize to analyzed WGS data of cfDNAs from the fresh and RT-7-days BM samples. Results show that, no matter which cfDNA, the z-score of each chromosome is within the normal diploid range (between -3 and 3)  . All theR > 0.65) of detectable miRNAs between BM cellular RNA and cell-free RNA . These data suggest that BM supernatant contains stable miRNAs whose abundance pattern is in accordance with miRNAs in BM cells and whose integrity can maintain pretty good even after BM is incubated at RT for 7 days. Moreover, abundance profiles of miRNAs in both cellular and cell-free RNAs show high similarity between different individuals.It is reported that miRNAs are present in human plasma in a remarkably stable form . Our resChanges of miRNA levels have been reported to be implicated in the initiation and progression of breast cancer , 21. HowOur study reports the abundant existence and outstanding stability of BM cfNA (cfDNA and cell-free miRNA) which mainly originated from breast-derived cells and contains genetic information similar to that in BM cells. These characteristics make BM cfNA a potential noninvasive sample for the study of breast cancer risk and early detection. Early screening for breast cancer has always been paid much attention. Over the past years, tests of ctNA in plasma have gained wide attention for cancer diagnosis and early detection –30. ctNACompared with that in plasma, cfNA in breast milk naturally has much more superiority in specificity and sensitivity of early breast cancer detection, because ctNA in blood would be extremely diluted by normal cfNA in circulation and constantly cleaned from the blood by the liver and kidney . So therOur data shows that patterns of miRNA expression and DNA methylation exhibit high similarity between BM samples from different mothers, which suggests a potential basal level exists in healthy women. Changes of miRNA levels and DNA methylation have been reported to be implicated in the initiation and progression of breast cancer , 32–34. Our study also raises questions about mechanisms of BM cfDNA and miRNA stability and cfNA releasing into BM from cells in vitro. The remarkable stability of cfDNA and miRNA in BM is very advantageous for long-distance transportation of BM samples. Research of the mechanism by which cfNAs keep intactness in BM could help us deeply understand this heterogeneous liquid and find enlightenment for the protection of cfNA samples. One possibility is that exosomes exist in BM and package cfNA inside like in plasma .Our results indicate that cfDNA with a major size of ~180 bp can be generated in vitro after incubation of BM at RT . The mosIdeal sample sources for detecting early nucleic acid changes are greatly desired to reduce the morbidity and mortality caused by breast cancer. We show here that cell-free DNA and microRNA exist in BM in a relatively stable form at RT; these cfNAs in BM mainly come from breast-derived cells and contain genetic information similar to that in BM cells. Perhaps, these characteristics might make BM cfNAs new noninvasive sample sources for searching early alterations of nucleic acid associated with the initiation and progression of breast cancer.


Action selection (AS), or selection of an action from a set of alternatives, is an important movement preparation process that engages a frontal-parietal network. The addition of AS demands to arm training after stroke could be used to engage this motor planning process and the neural network that supports it. The purpose of this case series is to describe the feasibility and outcomes associated with task-oriented arm training aimed at engaging the AS behavioral process and the related neural network in three individuals with chronic stroke. Three participants with mild to moderate motor deficits completed 13 to 15 sessions of task-oriented arm training that included AS cues for each movement repetition; cues dictated movement direction, height, or distance. Before and after training, individuals completed an AS brain-behavior probe during functional MRI. AS behavioral performance improved after training  in all participants while brain activation in the AS network  decreased in two participants. Gains in motor function were also found in all three participants, especially on patient-reported measures of perceived difficulty and confidence to complete upper extremity functional tasks. It was feasible to target the AS behavioral process and the related neural network through the addition of AS demands to functional, task-oriented arm training in three individuals with mild to moderate motor dysfunction poststroke. Successful performance of skilled motor tasks involves a variety of movement preparation processes in addition to movement execution. Action selection (AS), or selection of an action from a set of alternatives, is one of these movement preparation processes . The selTask-oriented training, the practice of goal-directed, functional tasks, is an important component of arm and hand rehabilitation after stroke –9. WhileThe addition of AS cues to task-oriented arm training may provide the opportunity to target the AS behavioral process and the brain regions that support this process within the context of functional movement training aimed at improving motor function. We chose to add AS demands to training as this movement preparation process is important in the performance of goal-direction actions , provideThree right-hand dominant individuals with chronic left hemisphere stroke were recruited from the local community. All three participants completed an AS brain-behavior probe to examine AS behavioral performance and brain activation with the contralesional right hand before and after a 3-week training phase. Clinical assessments of arm motor function were also completed before (two baseline assessments 1 week apart) and after training as well at a 3-week follow-up. Participants provided written informed consent according to a study protocol approved by the University of South Carolina Institutional Review Board (Protocol ID# Pro00032516).Individuals were eligible to participate if they were ≥18 years old, had a stroke at least 6 months prior to enrollment, were right-hand dominant  prior toA summary of initial presentation for the three participants is presented in The intervention involved 15 1.5-hour sessions of task-oriented training (5 times per week for 3 weeks) that focused on arm and hand function with a licensed physical therapist. Task-oriented arm training involves the repetitive practice of goal-directed, functional movements , 8. Any Action selection cues were added to every movement repetition during training. Cues were displayed on a screen directly in front of the participant using E-Prime 2.0.10  and were designed to require movement selection between right and left, close and far, or high and low ; Table 2Before and after the motor training period, participants completed an AS brain-behavior probe with the more impaired hand to examine AS behavioral performance and brain activation during AS. The behavioral task involved right or left movement of a joystick based on a visual cue in two different conditions. In the Select condition, the individual moved right or left based on an abstract rule . When a 3 isotropic voxels) and T2  structural scans for lesion identification and normalization of functional images. Diffusion weighted images  were acquired and used for the determination of corticospinal tract integrity.Before (Pre-TX) and after (Post-TX) the period of motor training, individuals completed the action selection behavioral task in a 3T Siemens Prisma MRI scanner with a 20-channel head coil and an MRI compatible joystick. Data from the MRI joystick was not available due to technical issues; therefore, task performance was quantified using data collected just prior to the MRI session. In a previous study using the same task in individuals post-stroke, the relative performance between the Select and Execute conditions was similar during functional MRI and practice in the lab just prior . Functio3 voxels. Images were then spatially smoothed with an isotropic Gaussian filter (FWHM 8 mm), and a temporal filter was applied (1/128 Hz) to remove low-frequency confounds. Data from each functional run were inspected for outliers due to excessive head motion (>2 mm translation or >0.2 radian rotation between volumes) or signal noise (Z > 5 from the mean image intensity) using the Artifact Detection Tool toolbox (http://www.nitrc.org/projects/artifact_detect); outliers were deweighted during statistical analysis.All functional imaging data were analyzed using SPM12 . First, the origin of the structural T1 image was checked and repositioned to the anterior commissure as needed. Volumes from each fMRI run were realigned and resliced to the first volume to account for motion artifact. Next, the realigned and resliced images were coregistered to the participant's structural T1-weighted image. The participant's structural image was normalized to a T1 older brain template with the stroke lesion masked out using the Clinical Toolbox in SPM . The norFirst-level statistical analysis was performed separately for each participant using a general linear model , 43. ForThe feasibility of task-oriented training that includes AS demands was examined through the assessment of the Select cue progression, Select accuracy, and total movement repetitions per session. Changes in AS task performance were assessed by performance on the AS brain-behavior probe while changes in the neural resources required to complete the AS task were assessed by the magnitude of brain activation in bilateral PMd, DLPFC, and parietal cortices.Changes in clinical measures of arm and hand function were also assessed and compared to published values for minimally clinically important difference (MCID), an indication that the change was meaningful, or minimal detectable change (MDC), an indication that the change was greater than measurement noise, for each measure if available. The three primary outcome measures, Action Research Arm Test (ARAT), Box & Blocks Test (BBT), and the Stroke Impact Scale (SIS) Hand Domain, were measured twice at baseline to establish stability of these measures prior to training, after training, and at a 3-week follow-up. The ARAT  and the All three participants tolerated task-oriented arm training that included AS demands. Individuals completed at least 13 training sessions . AS cue difficulty was progressed over sessions, individualized for each participants' accuracy during training . S2 and All three participants improved AS behavioral performance on the AS probe task from Pre-TX to Post-TX. Prior to training, as expected, accuracy was lower and RT was longer for the Select condition compared to the Execute condition , a measure of finger dexterity, after training . S1 alsoOverall, it was feasible to add AS demands to task-oriented arm training in three individuals with mild to moderate motor impairment due to stroke. After training, AS behavioral performance improved , and the neural resources utilized during AS decreased in bilateral premotor, prefrontal, and parietal cortices, suggesting the training paradigm was successful in targeting the AS behavioral process and its related neural network during training. Gains in motor function were also found in all three participants, especially in patient-reported measures of perceived difficulty and confidence, suggesting that motor function improvements were possible in the presence of the added action selection demands during training.All three participants showed improvement on the AS brain-behavior probe after training. Behavioral performance on the joystick-based AS task improved from pre- to posttraining  in all three participants while brain activation in key regions  decreased in two participants. Overall, these findings suggest that these individuals were able to perform the motor preparation process of AS more accurately and more quickly, utilizing less neural resource, after training. Overactivation during movement has frequently been reported in individuals post-stroke, both during simple movement execution tasks and in response to an increase in task demands , 52, 53.Gains in motor function were found despite the addition of AS demands to task-oriented training. Improvements in patient-reported outcome measures of perceived difficulty to complete hand tasks (SIS Hand domain) and confidence to complete upper extremity functional tasks (CAHM) were seen in all three individuals after training. Improvements in motor function were more variable, but all three participants improved performance on at least one measure . Patient-reported outcome measures may be more sensitive to deficits than performance outcome measures in individuals with mild motor impairment , althougTask-oriented training is a key aspect of arm rehabilitation after stroke. The principles of motor skill learning have been suggested to be a key factor in the implementation of task-oriented training after stroke , 55, howThis was a case series of three individuals with mild to moderate motor impairment in the chronic stage of stroke recovery. These three individuals tolerated the intervention and showed changes in AS performance and motor function. However, the feasibility and effectiveness of this approach beyond these three cases cannot be determined. Additionally, factors that may influence response to this training  are not possible in this case series. All three individuals received task-oriented arm training with AS demands. It is possible that the task-oriented training contributed to changes in AS behavioral performance and changes in brain activation during AS. Studies on the effect of task-oriented training on AS or motor planning are limited to a single small pilot study involving constraint-induced movement therapy . It is nIn conclusion, it was feasible to add AS demands to functional task-oriented arm training in three individuals with mild to moderate motor dysfunction poststroke. Improved AS behavioral performance was found after training that corresponded to a decrease in the neural resources required to complete an AS task. These gains in AS performance coincided with improvements in motor function suggesting that the addition of AS demands to training did not interfere with motor gains in these three individuals. The addition of AS demands to task-oriented arm training may provide an avenue to challenge motor preparation processes in addition to motor execution during functional training after stroke.


These Young Scientists’ Schools on bioinformatics have been organized every year since 2008 by the Institute of Cytology and Genetics of the Siberian Branch of the Russian Academy of Sciences and Novosibirsk State University [https://bmcbioinformatics.biomedcentral.com/articles/supplements/volume-21-supplement-11). The SBB Schools in Novosibirsk are satellite training meetings for young scientists and PhD students, which are organized either as satellite events for BGRS\SB (Bioinformatics of Genome Regulation and Structure \ Systems Biology) conferences series [This Special Issue of BMC Microbiology “Systems Biology and Bioinformatics” presents the materials discussed at the 11-th Young Scientists School “Systems Biology and Bioinformatics”-2019 (SBB-2019) in Novosibirsk, Russia  is a region with active hydrothermal activity, which contains outlets of unique natural hydrothermal petroleum  with disears old . The Uzoears old .The composition of the Uzon oil was investigated in several studies . Uzon oiIn this journal issue, Peltek et al.  analyzedActinomycetales (Actinobacteria). Typically, the geochemical parameters defined the structure and metabolic potential of microbial communities. This is confirmed by the genome sequence of Anoxybacillus flavithermus KU2–6-11 isolated from hot-spring in Uzon caldera, which was published recently [Aeribacillus bacteriophage AP45 was isolated from the Kamchatka [Geobacillus icigianus [In previous research, the majority of the studied oil sites contained representatives of recently . Other precently . The samrecently . While tamchatka  as well cigianus .Escherichia coli cells, which was published in BMC Microbiology [Going back to the BGRS post-conference issues, we would like to note fundamental systems biology work by late Dr. Vitaly A. Likhoshvai on the mathematical modeling of metabolic systems in obiology . This reobiology . Here itobiology .We aim to support international exchanges and education in bioinformatics and systems biology in the forms of the Schools for young scientists and make new results open keeping traditions of the BGRS conference series , 5, 20.


This case series illustrates clinical features and treatment outcomes of angioid streak associated CNV (AS-CNV) in 3 consecutive patients. Mean age of patients was 43.2 years with one female patient. Bilateral CNV was present in one patient. Comet-tail lesions were present in all cases. No underlying systemic association was found in any of the patients. All patients were treated with 3 loading doses of anti-VEGF injections (ranibizumab in two and aflibercept was used in one case). Subretinal fluid resolved in all cases with no recurrence of CNV activity at mean follow-up of 10.75 months. AS-CNV in Zambian eyes responds favourably to anti-VEGF injections. Angioid streaks (AS) were first described by Doyne as “irregular radial lines spreading from the optic nerve head to the retinal periphery” and represent visible irregular crack-like dehiscences in Bruch´s membrane that are associated with atrophic degeneration of the overlying retinal pigmented epithelium (RPE) -3. They Retrospective record collection of consecutive patients with AS-CNV reporting to a tertiary care eye centre in Lusaka, Zambia. Detailed ophthalmic examination at each visit was done. Informed consent was obtained from each patient for any interventional procedure performed as well as for data use.Three patients presented with AS-CNV between November 2015 to November 2018. The study adhered to the tenets of declaration of Helsinki. The clinical profiles of three patients are discussed in the following.Case 1: fourty two year old female presented with blurred vision in the left eye with best corrected visual acuity (BCVA) of 20/60 in left eye (LE) and 20/20 BCVA in right eye (RE). Anterior segment examination was normal. Fundus examination revealed AS radiating from the optic disc in both eyes. LE macula showed CNVM complex with subretinal haemorrhage. Mid-periphery of the retina showed comet-tail lesions in both eyes and more markedly at the posterior pole of RE. Fluorescein angiography showed the linear hyperfluorescent streaks radiating from the optic disc in both eyes. LE macula showed an early hyperfluorescent lesion roughly 1.5DD (disc diameter) with surrounding blocked fluorescence in the early phase, followed by increasing hyperfluorescence in the mid-phase and leakage in the late phase .This is the first case series from Zambia on treatment naïve AS-CNV describing their outcomes. Clinically none of the patients had any systemic association and in view of pathognomonic comet-tail lesions of PXE, their appearance in isolation without other features of PXE makes an interesting observation. This conforms to the revised criteria for diagnosing PXE where comet lesions constitute only minor eye criteria . BilaterThis series highlights the association comet-tail lesions in AS-CNV in the absence of clinical PXE, with favourable response of CNV in angioid streak to anti-VEGF ranibizumab/aflibercept with no recurrence. However, further longitudinal studies with a larger cohort and extended follow-up are required to know the long term behaviour of AS-CNV in Zambian eyes.Choroidal neovascularization (CNV) complicating AS  is an important cause of legal blindness and financial burden in middle-aged individuals affected by this condition;“Comet-tail” lesions are considered pathognomonic of PXE;The efficacy of anti-vascular endothelial growth factor (VEGF) agents, such as bevacizumab or ranibizumab, in the treatment of AS-associated CNV has been evaluated, with ranibizumab recently receiving official approval for this condition but there is still a high tendency of reactivation or recrudescence.This is the first series of AS-CNV from Zambia describing clinical profile and treatment outcomes; also it´s a well documented series of 4 eyes in African patients which to date is the largest number described;This series highlights presence of “Comet-tail” lesions in absence of PXE in African population;This case series shows good response of Anti-VEGF agents ranibizumab and aflibercept in AS-CNV with no incidence of reactivation; it establishes the potential role of aflibercept in AS-CNV which has been described in very limited studies so far; thus favourable outcomes can be expected in African eyes with AS-CNV on treatment with anti-VEGF agents.


While periprosthetic joint infections (PJIs) result in a small percentage of patients following arthroplasties, they are challenging to treat if they spread into bone and soft tissue. Treatment involves delivering antibiotics using poly(methyl methacrylate) (PMMA) bone cement. However, antibiotic release is insufficient for prolonged infections. Previous work demonstrated efficacy of incorporating insoluble cyclodextrin (CD) microparticles into PMMA to improve antibiotic release and allow for post-implantation drug refilling to occur in a tissue-mimicking model. To simulate how antibiotic refilling may be possible in more physiologically relevant models, this work investigated development of bone and muscle refilling models. The bone refilling model involved embedding PMMA-CD into rabbit femur and administering antibiotic via intraosseous infusion. Muscle tissue refilling model involved implanting PMMA-CD beads in bovine muscle tissue and administering antibiotic via tissue injection. Duration of antimicrobial activity of refilled PMMA-CD was evaluated. PMMA-CD composite in bone and muscle tissue models was capable of being refilled with antibiotics and resulted in prolonged antimicrobial activity. PMMA-CD provided sustained and on-demand antimicrobial activity without removal of implant if infection develops. Intraosseous infusion appeared to be a viable technique to enable refilling of PMMA-CD after implantation in bone, reporting for the first time the ability to refill PMMA in bone. Periprosthetic joint infections (PJIs) can be a devastating complication resulting from some arthroplasties and can involve infection of both the soft tissue surrounding the prosthetic implant as well as the inside of the bone in severe cases . To mitiTraditionally, to prepare antibiotic-laden PMMA, antibiotics such as aminoglycosides and glycopeptides are often incorporated into the PMMA during polymerization . HoweverIn an effort to improve upon the antibiotic release kinetics and to enable post-implantation antibiotic refilling to occur, a PMMA composite containing polymerized cyclodextrin (CD) microparticles was previously developed ,8. CycloTo simulate how antibiotic refilling of PMMA-CD composite embedded in femur could reasonably occur, a refilling model was developed in which PMMA-CD composite was embedded in the bone and IO infusion was used to provide direct access to the bone marrow to administer antibiotics and allow for refilling of embedded PMMA-CD. The antibiotic refilling ability of PMMA with and without CD microparticles embedded in femur was evaluated through post-refilling analysis of the depth of antibiotic diffusion into PMMA-CD composite and the resultant duration of antimicrobial activity. To translate the refilling model for the application of PMMA beads to treat muscle tissue PJIs, PMMA beads with and without CD microparticles were implanted in bovine muscle tissue and the tissue was directly injected with a bolus of antibiotic. Extent of refilling capacity of PMMA-CD composite beads was evaluated through administration of iterative antibiotic doses and subsequently investigating the resultant duration of antimicrobial activity of refilled PMMA-CD beads. To explore the possibility of refilling PMMA-CD composite beads through muscle tissue if they are initially covered in bacterial biofilm, a modified soft-tissue refilling model was developed where biofilms were formed on composite beads prior to implantation in the tissue. Duration of antimicrobial activity of refilled PMMA-CD composite beads with biofilm was evaluated and the remaining colony-forming unit counts on the bead and in the surrounding tissue were quantified after antibiotic treatment. Implementation of the proof-of-concept bone and muscle tissue refilling models developed in this work could provide insight into novel treatment options to improve outcomes for patients with chronic or prolonged PJIs.® HV (high viscosity) radiopaque bone cement  and Cook® Medical Intraosseous (IO) infusion needles   were purchased from eSutures . New Zealand white rabbit knee joint tissue was graciously donated by the Animal Resource Center (ARC) at Case Western Reserve University . Bovine abdominal muscle tissue was purchased from a local butcher . Green fluorescent protein (GFP)-labeled Staphylococcus aureus bacteria  was kindly provided by Dr. Edward Greenfield . All other reagents were purchased in the highest grade possible from Fisher Scientific .Lightly epichlorohydrin cross-linked β-CD pre-polymer (116 kDa molecular weight) was purchased from CycloLabs . Ethylene glycol diglycidyl ether was purchased from Polysciences Inc. . Rifampin (RMP) was purchased from Research Products International . SimplexInsoluble cyclodextrin (β-CD) microparticles were synthesized according to a previously published methodology ,8,25. Sp® HV surgical grade bone cement powder was mixed with 1 mL of methyl methacrylate monomer until a soft dough formed. For PMMA samples containing CD microparticles, 15 wt% CD microparticles (wt/wt) were added to each batch of PMMA prior to addition of liquid monomer and mixed until homogeneous. For the bone refilling model PMMA samples, the soft dough was directly finger-pressed and cured in the bone (displacing bone marrow) and had dimensions of approximately 6 mm tall and 4 mm diameter. For the muscle tissue refilling model PMMA samples, the soft dough was pressed into a thin layer (~2 mm thick) and small beads were punched out (6 mm diameter) and allowed to cure at room temperature. Properties of PMMA-CD composites have previously been characterized extensively including the ultimate compressive strength (~65 MPa) [For both bone and muscle tissue refilling models, PMMA was prepared using a similar methodology, according to manufacturer instructions. Specifically, 2-g samples of Simplex~65 MPa) , porosit~65 MPa) , and ant~65 MPa) .To simulate how we envisioned PMMA-CD composite antibiotic refilling in the scenario of treating arthroplasty PJIs, we set out to develop a PMMA-CD refilling model of the femur. Rabbit femurs were selected as an ideal ex vivo model, as they are frequently used in orthopedic in vivo models and rabbit bone mineral density has similarities to humans ,28. FemuIn an effort to evaluate the extent of antibiotic refilling possible with PMMA-CD composite when it was embedded in the femur, refilled PMMA-CD composite was explanted from the femur, sliced in half vertically, and analyzed using two metrics. First, stereomicroscope images were collected of the interior cut face of the PMMA-CD composite while it was still embedded in the bone and the front and back of the explanted PMMA-CD composite (with and without 15 wt% CD microparticles) to provide direct visualization of refilling. Depth or distance of diffusion of RMP refilling into PMMA-CD was directly quantified from the microscope images using ImageJ (2018) where up to eight measurements were collected on each half of the sample measured from the edge of the sample until the end of the visible “orange/red” region on the interior of PMMA-CD  and averaged.Staphylococcus aureus (S. aureus) bacteria [S. aureus culture was spread evenly on Trypticase soy agar petri dishes and the interior cut RMP refilled face of the PMMA-CD composite was placed face down on the surface of the agar. Dishes were incubated overnight at 37 °C and the following day the zone of inhibition was measured from the edge of the bacterial clearance to the edge of the PMMA-CD sample using calipers (four measurements per sample). PMMA-CD samples were transferred to a fresh S. aureus dish and this process was repeated until the zone of inhibition was no longer visible.Then, to determine the duration of antimicrobial activity possible from refilled PMMA-CD composite, explanted PMMA-CD samples were evaluated in a persistence zone of inhibition study against bacteria ,8. SpeciIn an effort to recapitulate the scenario in which antibiotic-filled PMMA-CD composite beads are used to treat PJIs, a PMMA-CD antibiotic refilling model was developed to mimic RMP refilling through bovine muscle. Small sections of muscle tissue (2 cm × 2 cm × 1 cm) were prepared, a small horizontal incision was made in the side (1 cm thick) of the tissue, and a PMMA-CD composite bead (with or without 15 wt% CD microparticles) was implanted. A small amount of cyanoacrylate glue was used to seal off the incision. The tissue containing the PMMA-CD composite bead was then embedded in 0.075% wt/vol agarose  in a six-well plate, as described previously . Once thS. aureus bacteria, according to a previously described methodology [Following 1–3 cycles of refilling, PMMA-CD composite beads were explanted from the muscle tissue and analyzed using two methods to determine the extent of antibiotic refilling possible in soft tissue. First, images of both the front and back of each PMMA-CD composite bead (with and without 15 wt% CD microparticles) were collected using a stereomicroscope to provide a direct visualization of the antibiotic refilling. Then, the duration of antimicrobial activity possible from each refilled PMMA-CD bead was determined through a persistence zone of inhibition study against hodology .S. aureus culture and incubated for 48 h at 37 °C. PMMA-CD beads were then lightly dried off and embedded into the muscle tissue sample and the tissue was held together with cyanoacrylate glue and embedded in 0.075% wt/vol agarose  in a six-well plate and allowed to solidify. Tissue was incubated for 24 h at 37 °C to allow for the infection to further develop. After this time, two 100-μL injections of RMP  were injected directly into the tissue at different locations ~6 mm away from the implanted PMMA-CD composite bead and the plate was covered and incubated at 37 °C for 48 h. As a control, a subset of tissues containing PMMA-CD composite (with and without 15 wt% CD microparticles) was not injected with any RMP to provide growth control counts of the S. aureus colony-forming units (CFUs) without the presence of drug . After 48 h of incubation with drug (or blank methanol control), tissue was removed from the agarose and the PMMA-CD composite bead was explanted and analyzed.To evaluate the possibility of allowing for antibiotic refilling to occur in soft tissue in the scenario in which a bacterial biofilm has formed on the surface of the implanted PMMA-CD composite bead, a modified muscle tissue infection antibiotic refilling model was developed. Prior to implantation in the muscle tissue sample (2 cm × 2 cm × 1 cm), PMMA-CD composite beads (with and without 15 wt% CD microparticles) were submerged in 1.5 mL of S. aureus [To evaluate the extent of antibiotic refilling possible in the presence of a bacterial biofilm on the surface of the PMMA-CD composite bead through muscle tissue, the explanted antibiotic refilled PMMA-CD beads were analyzed using two metrics. First, the extent of infection remaining in the tissue and on the surface of the PMMA-CD composite beads after treatment with RMP or methanol was analyzed through quantification of CFU counts. All the tissue surrounding the PMMA-CD composite beads was homogenized in 15 mL of 2× Trypticase soy broth. PMMA-CD composite beads were sonicated for 30 min in 3 mL of 2× Trypticase soy broth. For both solutions (from tissue and PMMA-CD composite beads), 70-μL aliquots of solution were plated out on Trypticase soy agar plates and incubated overnight at 37 °C. CFU counts were completed using ImageJ 2018). Extent of infection remaining after treatment with antibiotic was compared relative to the CFU count in control samples without any antibiotic. Separately, to analyze the duration of antimicrobial activity of PMMA-CD composite beads refilled with RMP through biofilm, refilled beads were explanted from the tissue and evaluated in a persistence zone of inhibition study against . Extent t-tests with unequal variances were conducted for depth of antibiotic diffusion (n = 4), persistence zone of inhibition (n = 3), and CFU counts (n = 3). The p-values of less than 0.05 were considered to be statistically significant .All data were reported as the mean and standard deviation of a minimum of 3–4 samples. Statistical analyses were carried out in Microsoft Excel 2016. Two-tailed Student’s p = 1.5 × 10−6).To quantify the relative amount of refilling possible in samples with and without CD microparticles using the IO infusion technique, stereomicroscope images were collected of the interior of all embedded PMMA-CD samples and ImageJ was used to measure the depth or distance of diffusion of RMP into PMMA-CD. This measurement provided insight regarding how much PMMA-CD material  was feasibly used in drug refilling and delivery functions. S. aureus. PMMA-CD samples were sliced in half and placed interior-side down on agar plates seeded with S. aureus. S. aureus for >14 days, whereas PMMA without CD microparticles (plain) was only able to clear S. aureus for <7 days. Over the first six days of the study, PMMA with 15 wt% CD microparticles had a significantly larger-sized zone of inhibition than the control, plain PMMA .To determine the duration of time in which RMP refilled PMMA-CD composites (with and without CD microparticles) were able to provide therapeutically relevant antimicrobial activity, refilled PMMA-CD samples were evaluated in a persistence zone of inhibition study against PMMA-CD beads with and without 15 wt% CD microparticles were implanted in the muscle tissue and refilled with either 1, 2, or 3 consecutive bolus injections of RMP. Following incubation with RMP over 48 h, PMMA-CD beads were explanted from the tissue and imaged using stereomicroscopy. S. aureus. p < 0.05).To evaluate the duration of antimicrobial activity possible from PMMA-CD samples refilled over three cycles, a persistence zone of inhibition study was carried out against S. aureus biofilms were statically formed on the surface of the PMMA-CD beads over 48 h prior to implantation in muscle tissue. Once implanted, the tissue was incubated for 24 h to allow for the infection to develop and either a bolus of RMP (dissolved in methanol) or blank methanol was injected directly into the muscle tissue and incubated for an additional 48 h. PMMA-CD beads were removed from the muscle tissue after this time and CFU counts were performed on both the PMMA-CD bead (via sonication) and the surrounding tissue (via homogenization).When biomaterials remain implanted in a patient over a prolonged period of time, they pose the risk of forming bacterial biofilms on their surface, particularly if they are being used in antimicrobial applications ,31,32. AS. aureus in a persistence zone of inhibition study  to be refilled with antibiotic (RMP) following implantation in bone and soft tissue for the treatment of chronic arthroplasty PJIs. Previous work has demonstrated the ability of PMMA-CD composite to be refilled in an agarose-based tissue phantom, but this model did not sufficiently replicate the properties of diffusion of drug through hard tissue, such as bone. To allow for appreciable antibiotic refilling to occur in the PMMA-CD composite, it is necessary that the PMMA-CD be in contact with a high concentration of antibiotic for a period of time . Since antibiotic cannot freely diffuse through bone, for the bone refilling model it was necessary to penetrate the bone and inject the drug directly into the bone marrow using IO infusion to get a high enough concentration of antibiotic in direct contact with the implanted PMMA-CD.Collectively, from the results of The extended duration of antimicrobial activity of the PMMA-CD composite implanted in bone refilled with IO infusion demonstrates the potential of the refilling model to provide the patient with an alternative treatment to PJIs where it may not be necessary to physically remove the infected arthroplasty implant, but simply locally administer more antibiotic as needed. Currently, if a PJI develops, the first line of treatment is systemic antibiotics followed by one- or two-stage revision procedures that result in significant trauma, mobility restrictions, and cost to the patient ,34. The Antimicrobial activity resulting from the plain PMMA refilled in the femur model was attributed to the small amount of RMP that passively diffused into the periphery of the PMMA, whereas the prolonged duration of antimicrobial activity that resulted from PMMA-CD composite was attributed to the greater amount of drug that was able to be refilled . While the bacterial load present on PMMA-CD beads and in surrounding muscle tissue after treatment with RMP was similar in PMMA with and without CD microparticles, composition of PMMA did have a dramatic impact on the amount of RMP filled into PMMA beads in the presence of infection see . As refiThe large error bars present in Conventional treatments for PJIs that have developed in the bone and muscle tissue surrounding the prosthetic implant involve radical measures such as months of systemic antibiotics and removal and replacement of the prosthetic, rendering the patient immobile for an extended period of time. The presented work demonstrated the ability of a PMMA-CD composite material to be refilled with antibiotics after being implanted in bone or muscle tissue. Refilled PMMA-CD had an extended duration of antimicrobial activity from 14–40+ days and PMMA-CD composites retained the ability to be refilled with antibiotics even in the presence of a biofilm and tissue infection. Of critical importance, this work demonstrated that PMMA-CD had the ability to provide sustained and on-demand antimicrobial therapy without the necessity to remove the implant if an infection develops. Furthermore, through this work, IO infusion appeared to be a viable, clinically used technique to enable refilling of PMMA-CD after implantation in bone, reporting for the first time the ability to refill PMMA in bone. In summary, PMMA-CD composite is a promising material that is versatile and can be applied to treat two aspects of PJIs—internal osteomyelitis and surrounding muscle tissue infections that can be refilled with antibiotics in a patient-customizable manner regardless of if it is implanted in bone or muscle tissue.


In this mSphere of Influence article, she reflects on how the paper “Efficient replication of Epstein-Barr virus in stratified epithelium  in vitro” by Temple et al.  has influenced her work on EBV molecular pathogenesis in the nasopharynx by highlighting the importance of using three-dimensional (3-D) culture models to study epithelial infection.Kathy Shair works in the field of Epstein-Barr virus (EBV)-associated cancers, with emphasis on nasopharyngeal carcinoma (NPC). In this mSphere of Influence article, she reflects on how the paper “Efficient replication of Epstein-Barr virus in stratified epithelium  Sample, her group uncovers what happens to de novo EBV infection upon differentiation of primary oral keratinocytes modeled in organotypic raft culture -associated cancers is that the EBV genome is not integrated, existing as multicopy episomes that favor latency instead of lytic infection. Many EBV studies related to epithelial infection are performed in two-dimensional 2-D) monolayer culture. Adherent monolayer cultures reproduce EBV latency but cannot recapitulate lytic reactivation in response to cellular differentiation—the physiological stimulus for EBV reactivation in epithelial cells –3. React–-D monola culture .de novo infection of primary keratinocytes is arguably the most intricate but likely the most authentic. The de novo infection conditions in primary organotypic rafts mirror the shuttling of EBV from B-cells to epithelial cells, reflecting EBV’s alternate cell tropism  surface of an organotypic raft that is undergoing differentiation. Several days later (4 to 6 days postinfection), the organotypic raft completes differentiation. Wounding of the raft is not necessary but encourages virus adsorption and provides access to the basal layer where latently infected cells (should they exist) are expected to reside  on metal grids or on commercial transwell membranes. There are other forms of 3-D culture, such as suspension in hydrogels or nonadherent culture dishes, but only ALI culture methods can generate self-organizing organoids that simulate the diversified cell types of the epithelium. Three-dimensional culture models are sophisticated, and establishing conditions for  tropism . What ma tropism  remarkabo reside  run the in vivo . There is a longstanding debate as to whether EBV and/or host genetics contributes to NPC risk, and epidemiology data would support that both are possible   . One int


Plasma cell disorders (PCDs) are identified in the clinical lab by detecting the monoclonal immunoglobulin (M-protein) which they produce. Traditionally, serum protein electrophoresis methods have been utilized to detect and isotype M-proteins. Increasing demands to detect low-level disease and new therapeutic monoclonal immunoglobulin treatments have stretched the electrophoretic methods to their analytical limits. Newer techniques based on mass spectrometry (MS) are emerging which have improved clinical and analytical performance. MS is gaining traction into clinical laboratories, and has replaced immunofixation electrophoresis (IFE) in routine practice at one institution. The International Myeloma Working Group (IMWG) Mass Spectrometry Committee reviewed the literature in order to summarize current data and to make recommendations regarding the role of mass spectrometric methods in diagnosing and monitoring patients with myeloma and related disorders. Current literature demonstrates that immune-enrichment of immunoglobulins coupled to intact light chain MALDI-TOF MS has clinical characteristics equivalent in performance to IFE with added benefits of detecting additional risk factors for PCDs, differentiating M-protein from therapeutic antibodies, and is a suitable replacement for IFE for diagnosing and monitoring multiple myeloma and related PCDs. In this paper we discuss the IMWG recommendations for the use of MS in PCDs. Central to the diagnosis and monitoring of most PCDs is detection of the monoclonal immunoglobulin components which are generally overproduced by the expanding plasma cell clone. This overproduced monoclonal immunoglobulin (often referred to as an M-protein or paraprotein) typically is an intact immunoglobulin, and also can be either the free light chain (LC) component alone or the heavy chain component alone in rare instances2.Plasma cell disorders (PCDs) are a group of diseases characterized by clonal expansion of plasma cells3. Serum protein electrophoresis (SPEP) enables the detection and relative quantitation of the M-protein, whereas serum immunofixation electrophoresis (IFE) enables establishment of M-protein isotype. Another widely utilized assay is the serum free light chain (sFLC) assay that utilizes specific antibodies for quantitation of circulating free kappa (κ) and lambda (λ) light chains (LCs)4. The sFLC assay is an analytically sensitive assay for M-protein detection whereby an abnormal κ/λ FLC ratio (κ/λ < 0.26 or >1.65) suggests the presence of an aberrant plasma or B-cell clone, but not all patients with multiple myeloma (MM) have abnormal sFLC ratios at diagnosis5. The sFLC assay has demonstrated a particularly important supportive role in the diagnosis, prognosis, and monitoring of PCDs, especially for amyloidosis and non-secretory MM patients7 In 2009, a panel of members of the IMWG met to develop guidelines for standard investigative workup of patients with suspected MM. The group evaluated studies that compared the diagnostic sensitivity and specificity of different combinations of testing panels and concluded that a combination of SPEP, serum IFE, and sFLC should be used for screening8. These protein studies have been incorporated in defining the various PCDs, as well as the uniform response criteria to assess treatment efficacy9.While the M-protein is homogeneous and typically constant in any particular patient, the heterogeneity of M-proteins from patient to patient is significant and thus a diverse set of methods are employed to characterize and detect M-proteins10 and their specific VDJH/DJH sequences by next-generation sequencing (NGS)11. These BM-based techniques are well-established and currently available for MRD testing after therapy. However, these MRD methods require an invasive procedure and a lab with a higher level of expertise to perform the testing. Attesting to its impact on prognosis, NGS is now an FDA-approved method for measuring MRD in MM. In addition, new monoclonal therapeutic antibodies (t-mAbs) designed to eradicate malignant plasma cells are producing interferences making it difficult to distinguish between a patient’s M-protein and the t-mAb drug on electrophoretic methods12.While the clinical utility of the electrophoretic methods to screen and monitor PCDs has been well-established, several changes in the treatment of PCDs are pushing these methods to their analytical limits. Dramatic improvement in the treatment response of MM patients to new chemo and immune therapies is challenging long-held assumptions about this ominous disease. In particular, as durable remissions can now be achieved for most MM patients, highly sensitive methods capable of detecting low-level disease are important for patient counseling. The long-standing routine serum electrophoretic methods (SPEP and IFE) are not capable of providing the analytical sensitivity needed to assess minimal residual disease (MRD). As a result, laboratories have turned to bone marrow (BM) aspirates and/or biopsies and detecting residual malignant plasma cells with high-sensitivity flow cytometryA search for a more convenient serum-based test to complement BM MRD detection and aid in resolving t-mAb interferences was sought to address limitations in traditional testing. Mass spectrometry (MS) is aptly suited for this task as the improvements in instrumentation and techniques have resulted in increased resolution and sensitivity which have outpaced improvements in electrophoresis.13. Each plasma cell produces a unique Ig with a specific CDR due to the adaptive immune system’s optimization of the CDR via somatic hypermutation to increase the affinity of the Ig to its target antigen. The resulting CDR amino acid sequence is unique, and each plasma cell clone has a different peptide sequence and overall mass, which is the basis of the M-protein detection by MS14. Two MS methods have emerged in the literature. Both methods start with immune-enrichment of patient immunoglobulins (Igs) but differ on the analytical target used to detect the M-protein. One method utilizes Ig trypsin digestion and detection of peptides specific to the M-protein CDR18. This method has been termed the “clonotypic peptide” approach. The second method utilizes total LC mass distributions from Igs which have been chemically reduced and denatured into heavy and light chain components20. This method will be termed intact LC mass measurements herein.The basis of all the mass spectrometric methods for detecting M-proteins is the unique sequence of the antigen binding region, also called the complementarity determining region (CDR) of the Ig15, assess for MRD in complete response (CR) patients21, and to differentiate M-proteins from Daratumumab17. The clonotypic method is very analytically sensitive with M-protein detection rates down to 0.001 g/L giving it potential to be a serum-based MRD method16. However, the method has some limitations that hinder it from being suitable for all patients. In some patients with M-proteins above 10 g/L, only constant/framework peptides were detected making it impossible to follow or detect M-proteins in these patients. In addition, as the CDR also contains framework regions, which have less diversity across the polyclonal background, clonotypic peptide selection needs to be done judiciously to assure the uniqueness of the clonotypic peptide to the M-protein. As such, the success of identifying a clonotypic peptide still requires genetic CDR sequence information from the malignant plasma cells. Once a potential clonotypic peptide is determined, a search of the human proteome is necessary to assure its uniqueness. Unfortunately, a complete library of the possible human CDR tryptic peptides is not yet available. All this requires very thorough understanding of immunoglobulin CDR region to assure definitive test results. Lastly, screening for M-proteins using the clonotypic peptide method has not been demonstrated. Recent work utilizing IFE gels followed by the clonotypic approach has aided in simplifying the pre-analytical work for the clonotypic method22, but does not remove IFE from its screening role.The clonotypic peptide approach has been employed to monitor MM patientsMore clinical data is still needed to determine if the MRD positivity by the clonotypic approach translates into disease-free and overall survival benefits. While most studies utilizing the clonotypic method detect the presence of the M-protein in samples from patients who are BM MRD-negative, the clinical implications of this will require more time to elucidate.23. The first version of this intact LC mass measurement method was performed on higher performance mass spectrometers (ESI-Q-TOFs) with liquid chromatography. This method  was found to have an analytical limit of quantitation of 0.05 g/L and a limit of detection of 0.01 g/L. Later this method was adapted to matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry (MS)24, which eliminated the chromatography step and reduced analytical time from 20 min to 10 s. Eventually, the MALDI-TOF method was modified to include immuno-enrichments to conclusively identify the M-protein isotype in a similar fashion to IFE26. The discussion and recommendations for MS contained in this working group report, unless otherwise specified, pertain to this intact LC MALDI-TOF MS method.A second more simpler and practical approach for M-protein detection relies on scanning the overall mass distribution of denatured intact Ig LCs. In this method, the polyclonal Ig LC mass distribution results in two normal distributions, one for the lambda and second for the kappa LCs27. At this point in time, over 40,000 clinical samples have been analyzed by the intact LC MALDI-TOF method at Mayo Clinic. The Mass-Fix assay has been found to be comparable to IFE in terms of assay turnaround time and ease of interpretation with the added benefit of reduced labor. These features make the intact LC method more economically attractive in comparison to the clonotypic method. Commercial efforts are being made to automate the technique and provide a high-throughput method which would be widely accessible for routine laboratory implementation. Validation is currently being performed in several clinical laboratories.The intact LC MALDI-TOF MS assay is referred to as Mass-Fix at the Mayo Clinic to emphasize its similarity to conventional immunofixation. It has been clinically validated as a lab-developed test at the Mayo Clinic25. The ability of intact LC MALDI-TOF MS method to detect M-proteins was retrospectively evaluated on a large cohort of patient samples with physician-ordered SPEP and IFE, which included a wide variety of M-proteins encountered in clinical practice. MALDI-TOF MS detected M-proteins in 100% of samples that were positive by both SPEP and IFE (n = 84) and in 97% of samples positive by IFE but negative by SPEP (68 of 70). In samples negative by both SPEP and IFE (n = 28), MALDI-TOF identified one positive case20. To test analytical sensitivity, a cohort of 27 PCD patients was diluted into normal human serum and both IFE and intact LC MALDI-TOF were performed. The percentage of M-proteins detected by the intact LC MALDI-TOF was significantly higher than IFE at every dilution. A second prospective study involving 290 patients with documented PCDs demonstrated that substitution of serum and urine intact LC MALDI-TOF testing results for convention IFE results allowed for detection of disease in 18 more patients. Overall, MS had higher clinical sensitivity than IFE in detecting M-proteins in patients with abnormal FLC ratios. A combination of FLC, serum and urine Mass-Fix performed equally as well as current IMWG recommendations of PEL, IFE, and sFLC28. Incorporation of serum FLC MALDI-TOF MS26 has been shown to improve the sensitivity of M-protein detection during monitoring of AL amyloidosis30. The intact LC method has also been used for the diagnosis of heavy chain diseases31.Several comparisons of the intact LC MALDI-TOF MS and IFE have been published32 who were initially negative for a PCD by SPEP but who were found to have a PCD on later follow-up were studied. Since the initial screen was performed by SPEP, the study assessed the ability of IFE, the intact LC MALDI-TOF method (Mass-Fix), as well as a more sensitive miRAMM method to detect the M-protein in the original sample. M-protein would have been detected in 11%, 50%, and 67% of these participants if initial screening was performed by IFE, MALDI-TOF, and miRAMM, respectively33. A preliminary study of the pre-commercial intact LC MALDI-TOF method  utilizing samples from the GEM-CESAT trial patients demonstrated moderate correlation with MRD assessment by next-generation flow MRD status34.A more recent study confirmed the increased sensitivity and accuracy of intact LC MALDI-TOF MS over IFE. In this study, 226 patients from the Olmsted screening cohort35. The inability to discriminate between daratumumab and M-protein was most likely due to the lower resolution of the MALDI-TOF assay, as studies involving the higher resolution LC-ESI-QTOF (miRAMM) MS had a 100% success rate of differentiating the t-mAb  from M-protein36.The intact LC method MS can give useful information to discriminate between t-mAbs and M-proteins as the mass of the t-mAb LC can be measured. Using the intact LC MALDI-TOF method, daratumumab and IgG kappa M-proteins were distinguishable in 84% of the samples tested28, Additional studies demonstrated that these LCs contained N-linked glycosylation and were to be more prevalent in patients with AL-amyloidosis37 and cold agglutinin disease38 than other PCDs. Interestingly, LC glycosylation was also found to be an independent risk factor for the progression of MGUS to myeloma and related disorders39. These glycosylated LCs were demonstrated to be present years before the diagnosis of myeloma of related disorder and hence may allow for identification of patients at higher risk for PCDs40.A previously underappreciated finding of M-proteins when employing the intact LC method was the observation of M-proteins with a LC mass that was broad and outside the normal mass ranges of kappa and lambda LCs−4 to 10−5 by 6 color flow cytometry) in 80% (n = 16) and 60% (n = 25) of patients at 6–12 months, respectively41. The influence of Ig half-life on the MRD data needs to be further elucidated. However, since miRAMM MS is a serum-based assay, the test could be performed serially allowing the rate of response to be assessed over time. A second more recent study also compared the intact LC MALDI-TOF MS with BM flow cytometry-based MRD42. This exploratory study demonstrated MALDI‐TOF MS was concordant with MRD flow for 44/71 (62%) patients (p = 0.342). Amongst the 27 discordant cases, 17 were detectable only by MALDI-TOF MS, and 10 detectable only by MRD flow. Additional prospective studies are required to delineate the role and timing of these modalities in assessing MRD.MS using the more sensitive miRAMM intact LC approach may have a role in MRD detection. In one study, residual monoclonal proteins were detected in patients who were in stringent CR (sCR) (1020. As summarized in Table We conclude that MS has the advantage of increased accuracy, documented clinical and analytic sensitivity, and the intact LC MALDI-TOF method is easier on laboratory work flow for the detection of M-proteinsAJR Checklist.


Researchers have observed the free-propagation of a single microcavity polariton directly and its self-interference when scattering upon a defect. These experimental observations of quantum hydrodynamics in the single polariton limit test the wave-particle duality and aid in the development of polariton-based photonic circuits in quantum information processing. Microcavity exciton-polaritons, especially those in the mesoscopic solid-state platform and consistently examined12, have unique potential in this domain, including strong exciton and photon coupling resulting in the formation of vacuum Rabi polaritons. To date, the quantum fluid dynamics of a single polariton have rarely been examined.Quantum information processing requires the robust generation and manipulation of single and few quanta in a scalable platform with long coherence and exceptional quantum state preparation and measurement. In the domain of a photonic qubit, control of a single photon is canonically based on multiplexed linear optical elements or nonlinear electro-optic circuitsLight: Science & Applications, Suárez-Forero et al.13 realized the conversion of single photons from a semiconductor quantum dot into propagating microcavity polaritons and subsequently witnessed the unique self-interference of single polaritons when scattered across a structural defect. The textbook model of a cavity-quantum emitter system is characterized by the exciton–photon coupling or Rabi interaction rate g, the quantum emitter decay rate γ (in both spontaneous emission and dephasing), and the cavity decay rate κ. The strong coupling regime, where g) in its momentum-energy dispersion diagram, resulting in the anti-crossing of the well-known upper and lower polariton branches. By mapping the polariton dispersion and meticulously optimizing the external quantum dot size and single-photon injection angle, Suárez-Forero et al. managed to couple the single excitonic emission from the quantum dot, verified by second-order correlation measurements, near the lower polariton branch into the cavity-quantum well system, consequently generating single polaritons. The polariton population was estimated by counting the photons emitted from the cavity and the polariton lifetime estimated by fitting the observed energy dispersion map. The researchers further observed a real-space propagation up to 400 μm of single polaritons inside the cavity owing to the in-plane momentum acquired during the excitation process. The fact that the observed propagation is absent when the quantum dot exciton is tuned such that it is off-resonant with the polariton dispersion further fortifies the authors’ hypothesis13.Reporting in To fully capture the quantum hydrodynamical behavior of a polariton during its propagation from the single-photon injection region to the defect scattering region, Suárez-Forero et al. etched the substrate of the cavity-quantum well to realize an optimal single polariton propagation map via transmission measurements. The single-photon emission rate from the quantum dot was also enhanced by embedding the quantum dot into a low-quality-factor distributed Bragg reflector cavity and pumping with a 320 MHz repetition rate laser for maximum possible single-photon stream  injection into the cavity-quantum well system. Through this transmission configuration, the researchers unveiled the particular interference pattern of the single polariton due to scattering by a structural defect. By comparing the single-photon injection rate and the obtained polariton lifetime, the researchers convincingly demonstrated that they access the nonclassical regime with only one single polariton present in the cavity, which strongly indicates that the observed interference pattern was not caused by subsequent polaritons. Instead, it was a result of the self-interference of a single polariton, with the interference pattern generated by integrating each scattering event of a single polariton impinging on the defect (blue circle), as illustrated in Fig. This observation of the self-interference of a single polariton is a direct signature of the wave-particle duality of single particles in textbook quantum mechanics. The observed fringes ahead of the defect reveal the polariton quantum mechanical nature, which cannot be explained by classic approaches. Indeed, this experiment with a full spatial mapping of the electromagnetic field provides an alternative testbed for Wheeler’s delayed-choice experiment, the violation of causal time-ordering, and the study of nonlocal effects. In terms of applications, the cavity mesoscopic system used in this work provides insights into single polariton devices and is thus an important step towards polariton-based integrated quantum photonic circuits.


Drosophila egg chamber elongation. First, egg chamber rotation facilitates the dorsal-ventral alignment of the extracellular matrix and of the cell basal actin fibers. Both fiber-like structures form supracellular networks constraining the egg growth in a polarized fashion thus working as ‘molecular corsets’. Second, the supracellular actin fiber network, powered by myosin periodic oscillation, contracts anisotropically driving tissue extension along the egg anterior-posterior axis. During both processes, cellular and supracellular planar polarity provide a critical cue to control Drosophila egg chamber elongation. Here we review how different planar polarized networks are built, maintained and function at both cellular and supracellular levels in the Drosophila ovarian epithelium.Tissue elongation is known to be controlled by oriented cell division, elongation, migration and rearrangement. While these cellular processes have been extensively studied, new emerging supracellular mechanisms driving tissue extension have recently been unveiled. Tissue rotation and actomyosin contractions have been shown to be key processes driving  Tissue extension is a fundamental process during embryo development. The extension of tissues contributes to shape the developing embryo and drives the separation of groups of cells that will form different parts of an animal. Unraveling the mechanisms that drive tissue extension is key to understand how life emerges from a cluster of cells. A key and archetypal tissue elongation process is the one directed along the anterior-posterior (AP) axis of the embryo that drives the separation of the head region from the posterior region where the future brain and the anus of the animal will eventually form, respectively . TherefoDrosophila egg chamber elongation has been revealed: supracellular networks, emerging from the coupling of local fiber meshworks and controlled by subcellular planar cell polarity, form around the egg and function as a mechanical corset directing AP egg elongation  and planar cell polarity (PCP). ABP is established along the cell apical-basal axis and is necessary to coordinate cell-cell interaction forming the epithelial barriers that separate the inner from the outer side . PCP is and wing . In addiuide PCP . This de pathway . In this pathway .Drosophila oogenesis, the egg undergoes maturation before being expelled from the ovary and laid by the mother. The Drosophila ovary is composed of 15–20 ovarioles  motor protein to form an active contracting scaffold that directs egg extension . How areBetween S2 and S8, the egg chamber rotates along the AP axis . Since sWhat are the cellular mechanisms and the signaling factors controlling the directed collective movement of follicle cells? Actin-based protrusions, formed at the follicle leading edge, often can function as exploratory antennas or traction force generators directing cell movement . Both fiActin bundles at the basal side of follicle cells align orthogonally to the AP axis from very early stages before egg chamber formation and during germarium development. This pre-polarized network could function as a cue to initiate directed follicle cell migration. While egg chamber rotation is thus the original cause of ECM fibril but not actin bundle polarity, rotation was shown to be responsible for the maintenance of actin bundle polarity during egg chamber maturation .Egg chamber rotation stops at early stage 9, when the oscillatory contraction of the follicle basal actomyosin network starts to guide the second phase of egg chamber extension.Drosophila embryo gastrulation where cell-cell concerted net accumulation of MyoII ensures rapid and progressive cell apical surface reduction . This shows that the egg chamber during S9-S10 is under anisotropic tension with higher tension along the DV axis . Both inHow is stress-fiber polarity established and maintained? F-actin stress-fibers align along the DV axis at very early stages of oogenesis (region 2b) under the control of an unknown factor. Stress-fiber alignment is eventually maintained during S9-S10 under the control of cell-cell adhesion and Cdc42 . When E-Cell-ECM adhesions could also play a role in the organization of stress-fibers at the basal side of follicle cells since the inhibition of cell-matrix adhesion by RNAi, optogenetics, or loss-off-function alleles can eventually affect F-actin and MyoII levels and/or distribution in the basal region of follicle cells . FurtherThe spatial pattern of MyoII oscillations can be divided into three phases: (1) an initiation phase during early S9 (ES9) when MyoII oscillations appear in the anterior 1/3 region of the egg chamber; (2) a propagation phase between middle S9 (MS9) and beginning of S10 (S10A) when MyoII oscillations spread by shifting posteriorly and increasing in amplitude; and (3) a stabilization phase during middle to late of S10 (S10B) when all follicle cells contacting the oocyte accumulate medial-basal MyoII and MyoII oscillations reduce in amplitude maintaining high intensity levels. How the MyoII spatiotemporal pattern is controlled is still poorly understood. Drosophila egg chamber, a powerful model system to study the cellular mechanisms driving epithelial morphogenesis.Life often starts from a shapeless cluster of cells. Tissue elongation is among the very first morphogenetic processes to mold this cellular cluster during embryo development. Studying the fundamental mechanisms driving epithelial extension is thus a very exciting opportunity that will help us understand the emergence of life. In this review, we focus on the process of elongation in the Over the last decade, a new mechanism driving tissue elongation has been unraveled: polarized forces, driven by a molecular corset, work to extend the egg chamber. The molecular corset is a two-tier wafer-like scaffold: F-actin stress-fibers at the basal side of follicle cells underlay polarized ECM fibrils at the BM. Both fiber networks are DV polarized and are wrapped around the egg chamber. While during the first phase of elongation (until S8) the corset works as a passive scaffold, during the second phase (S9-S10) the corset generates active forces powered by actomyosin contractions. F-actin stress-fibers are pre-polarized in the germarium, while ECM fibril polarization is under the control of egg chamber rotation around the AP axis during S2 to S8. Egg chamber rotation is driven by follicle cell collective migration: follicle cells undergo rounds of revolution propelled by polarized lamellipodia. The ECM secreted by follicle cells during migration results in the formation of DV aligned fibrils. Which mechanism controls the direction of follicle cell collective migration? Egg chambers are connected to each other via stalk cells at the anterior and posterior poles forming a pearl-string-like structure. These polar connecting sites could bias the direction of follicle cell migration by limiting the egg-chamber free degrees of rotation. If the direction of migration is imposed by the boundary conditions of the system, it is still not clear which mechanism controls the sense of egg chamber rotation. Egg chambers belonging to the same ‘string’ can rotate both in a clockwise and anti-clockwise direction . Under tEgg chambers rotate with equal angular velocity during S3 to S7. Constant angular velocity at different egg-chamber stages can be achieved if follicle cells finely tune their speed of migration in a way that is directly proportional to the egg-chamber radius. Strong mechanical coupling between egg chambers belonging to the same ‘string’ could explain how angular velocity is conserved between egg chambers of different sizes: the rotation of one egg chamber would impose the same angular velocity to the other neighboring egg chambers (like wheels of different sizes mounted on the same axle). Nevertheless, this hypothesis may be ruled out since egg chambers on the same string may rotate in opposite directions. Future work is necessary to unveil the mechanisms controlling follicle cell migration imposing constant angular velocity to egg chambers at different stages.The egg chamber is an ovoid structure. If all cells of one egg chamber migrate around the AP axis with the same angular velocity, cells located at the poles must migrate at slower speed than cells at the equator. How is cell speed finely controlled at different egg-chamber AP positions? Follicle cell collective migration could result from a cell leader-follower based mechanism : for insStalk cells form connecting regions between egg chambers. If neighboring egg chambers rotate in the opposite sense, stalks cells would experience torsion opposing resistance to rotation. Cell intercalation between stalk cells and follicle cells at the egg-chamber poles is a mechanism that could facilitate counter-rotation between neighboring egg chambers. Future work is necessary to better elucidate the role of stalks during egg chamber rotation.The periodic contraction of the cell actomyosin cytoskeleton is a process usually reported during cell shape changes and tissue morphogenesis . The bioXW and AP prepared the figures. MR commented on the figure revision. All authors wrote and revised 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.


Transcutaneous aortic valve replacement (TAVR) has become a widely accepted minimally invasive approach for treatment of severe aortic stenosis. Self-expandable prostheses are commonly the device of choice, with excellent procedural success and durability. However, there have been several recent case reports of infolding of the self-expandable prosthesis during development with subsequent malfunction and need for further intervention. We present a case of self-expandable valve prosthesis infolding managed by balloon postdilation, and summarize the cases reported in the literature to date in an attempt to increase awareness of this serious technical problem and the factors associated with it. Transcatheter aortic valve replacement (TAVR) is now a well-established therapy for patients with symptomatic severe aortic stenosis. It has been proven in randomized controlled studies to be non-inferior compared with surgical aortic valve replacement (SAVR) among patients with low, intermediate, and high surgical risk -4. The m2, and a mean gradient of 52 mmhg; there was also moderate aortic insufficiency. Cardiac computed tomography demonstrated heavily calcified valve with estimated annulus perimeter of 86 mm consistent with a 34-mm Evolut R  valve . Transthoracic echocardiogram revealed an ejection fraction of 35%, aortic valve area estimated at 0.5 cme Figure .The Evolut R was advanced through in-line sheath in the right transfemoral access. First deployment at the two-thirds position demonstrated a high implant. The valve was recaptured and redeployed to optimal depth but the inflow part of the valve appeared to be under-expanded and with the appearance of a vertical line in the axis of the valve; this was persistent after the valve was fully released Figure . TransesPost-dilation was performed initially with 27-mm balloon with disappearance of the distortion Video .Repeat TEE images revealed only trace paravalvular leak.Several cases of infolding of self-expandable valves have been reported in the literature -11. The The most common clinical scenario for occurrence of infolding is in the patients who require large valve sizes (29 mm or larger) and in combination with significant valve calcifications. Careful fluoroscopic inspection, sometimes rotational, of the valve could help detect this serious complication [In Table Infolding of self-expandable valves is an underrecognized complication of TAVR by the structural interventionalists that is associated with several anatomical and device-related factors. There is need for more research and data collection to further evaluate the best approach to manage it and whether it is associated with any adverse clinical events.