We conclude that epigenetic modifications can influence the sex ratio variation in zebrafish families and may generate skewed sex ratios, which could have a negative consequence for population fitness in species with genotype-environment interaction sex-determining system under rapid environmental changes.The plasticity of sexual phenotype in response to environmental conditions results in biased sex ratios, and their variation has an effect on population dynamics. Epigenetic modifications can modulate sex ratio variation in species, where sex is determined by genetic and environmental factors. However, the role of epigenetic mechanisms underlying skewed sex ratios is far from being clear and is still an object of debate in evolutionary developmental biology. In this study, we used zebrafish as a model animal to investigate the effect of DNA methylation on sex ratio variation in sex-biased families in response to environmental temperature. Two sex-biased families with a significant difference in sex ratio were selected for genome-wide DNA methylation analysis using reduced representation bisulfite sequencing (RRBS). The results showed significant genome-wide methylation differences between male-biased and female-biased families, with a greater number of methylated CpG sites in testes than ovaries. Likewise, pronounced differences between testes and ovaries were identified within both families, where the male-biased family exhibited a higher number of methylated sites than the female-biased family. The effect of temperature showed more methylated positions in the high incubation temperature than the control temperature. We found differential methylation of many reproduction-related genes (e.g.,  In evolutionary developmental biology, the mechanism of sex determination is one of the most important developmental processes in bipotential gonad to differentiate into a testis or an ovary and establish the sex of an organism in a binary fate decision. Gonochoristic vertebrates exhibit two major types of sex determination, namely genotypic sex determination (GSD) and environmental sex determination (ESD). In organisms with GSD, sex is determined at the time of fertilization by inheritance of genetic factors, whereas in organisms with ESD, sex is determined after fertilization in response to environmental conditions . HoweverDanio rerio), a small tropical fish, is an excellent research model animal to study the mechanism of sex determination and the effect of environmental changes on gonadal differentiation in vertebrates , after the yolk sac was absorbed. At this stage, the larvae of each family were transferred in 7-L tanks. High rearing density during gonadal differentiation in zebrafish  control group was kept at a constant temperature of 28°C, the standard rearing temperature for zebrafish , throughStatistical analysis of the sex ratio was performed using the linear logistic model with a binary response variable by applying the GLIMMIX procedure of SAS System 9.3 . To chart-test procedure by inclusion of the PDIFF option in the Lsmeans statement.To select the families for reduced representation bisulfite sequencing (RRBS) analysis in this study, two decision criteria were applied: 1) a high difference in the proportion of males between the families under control condition (model 1), and 2) a high difference in the proportion of males between control and treatment groups within the families. The statistical analysis for differences in the proportion of males with regard to the classification of sex-biased families was carried out for the two selected families  considering the fixed effect of family, treatment, and family-treatment interaction using the following model 2:We collected the gonadal tissue samples of 6 females and 12 males in each experimental group: control male, control female, treatment male, and treatment female, in two different zebrafish families  separately. All animals were at the same age , when the samples were taken. Due to the small size of the male gonad tissue, we collected more male gonad samples to pool the DNA samples of two males in each group in the next step of the laboratory analysis. To this end, a total number of 72 gonad tissue samples from male and female animals were collected for RRBS analysis in this study . For thiGenomic DNA was extracted from gonad samples using the AllPrep DNA/RNA Mini Kit (Qiagen) with proteinase K and RNase A treatment according to the manufacturer’s recommendation. About 20 mg of the ovary tissue from each individual was used for DNA extraction, while the pooled testes of two animals were used to recover enough DNA. The integrity of intact DNA was assessed on 1.2% agarose gel electrophoresis and the quantity was determined using a NanoDrop spectrophotometer. Two micrograms of DNA sample were spiked-in with 1% DNA control [unmethylated cl857 Sam7 Lambda DNA (Promega)] and digested overnight with 100U MspI (New England Biolabs). The DNA fragments were recovered, end-repaired, A-tailed and ligated to TruSeq DNA adapters (cytosines methylated) using the TruSeq Nano DNA library prep kit (Illumina) according to the manufacturer’s protocols. The adapter-ligated DNA libraries were subjected to bisulfite conversion using the EZ DNA Methylation-Gold Kit according to the manufacturer’s protocols (Zymo Research). The libraries were amplified after bisulfite conversion using the PfuTurbo Cx Hotstart DNA Polymerase kit (Stratagene). The quality of the libraries was assessed on 2100 Bioanalyzer using a high-sensitive DNA chip (Agilent Technologies). The normalized RRBS libraries were multiplexed and parallel sequenced for 121 bp single reads on the HiSeq 2500 system (Illumina) at the sequencing facility of Research Institute for Farm Animal Biology (FBN), Dummerstorf, Germany according to the manufacturer’s recommendations using the HiSeq SR Cluster and HiSeq SBS Kit v4 (Illumina).Danio rerio reference genome (GRCz11) downloaded from Ensembl (http://ftp.ensembl.org/pub/release-100/fasta/danio_rerio/dna/) and performed methylation calls using Bismark version 0.22.3 (https://www.bioinformatics.babraham.ac.uk/projects/bismark/). Visualization of mapped data was performed using SeqMonk (https://www.bioinformatics.babraham.ac.uk/projects/seqmonk/).The bcl2fastq2 conversion software v2.19 was used to convert base call (BCL) files into fastq files. The overall quality of sequencing reads was inspected pre- and post-processing using FastQC . The raw fastq files were pre-processed to remove adapter-like sequence and trim 2 bp artificial filled-in of the blunt-end of both 5′and 3′ using TrimGalore (v.0.6.5). Cleaned reads were mapped to the 2 fold change. To fit the total read count at each genomic CpG site, the generalized linear approach modeled the proportion of methylated reads as an over-dispersed binomial distribution. This method allows direct modeling of the inherent variability of the data and thus possibly more realistic analysis of the data. The CpG sites with low coverage (less than 8) were removed from the downstream analysis based on the quality control proposed by edgeR to avoid unreliable results of methylation levels across all samples  were mapped at least in one sample. The raw CpGs were filtered to keep only CpG sites that were covered at least 10 reads and present in all samples. Finally, only 210,009 CpG sites passed the filters and used in downstream analyses. The RRBS analysis was performed in the R-Statistics program  using th samples . The sta samples  was appl samples , in whic samples . This lip-values less than 0.05 were investigated to be enriched in gene set enrichment analysis. All functional enrichment analyses were performed in R-Statistics program . Considering only the families in the control group  to determine the sex-biased families, and applying a logistic model, there was a significant effect of the factor of family  and a significant difference in the proportion of males between the two selected families (p < 0.0015). Considering only the families in the treatment group (model 1), there was also a significant effect of the factor family  and a significant difference in the proportion of males in the treatment group between the two selected families (p < 0.006). In the statistical analysis considering only the two selected families , there was a significant difference in the proportion of males independent of temperature effect . Using the same model for control condition, there was also a significant difference in the proportion of males in the two selected families (40.74 vs. 70.31), which is important in terms of it being a sex-biased family (p = 0.0017) (p = 0.0184) across families. However, the significance level between control and treatment groups was not very pronounced within the two selected families (p = 0.0590 and p = 0.1454). Since all animals used in this study were kept under the same husbandry conditions and recirculating system with an appropriate stocking density , the effect of tank on the level of methylation and consequently on the sex ratio variation within families were not presented.In this study, the effect of high incubation temperature (35°C) during embryonic development from 5 to 24 hpf compared to the control temperature  on sex ratio variation of different zebrafish families (N = 17 families) was examined. In the rest of the paper, the terms “treatment” and “control” are used instead of high incubation temperature and control temperature for simplicity. The results showed a wide range of sex ratio variation between families , suggest 0.0017) . ConsideTo explore the effect of epigenetic mechanisms on sexual phenotype of sex-biased zebrafish families, we performed multidimensional scaling analysis (MDS) of RRBS datasets to visualize overall differences in DNA methylation  between individual samples of different experimental groups . The M-vWe further considered the methylation pattern in the context of annotated genes and CpG islands . The varTo assess the differences in DNA methylation of sex-biased zebrafish families, given the clear clustering pattern in The overall results of differentially methylated CpG sites (DMCs) showed that the male-biased family exhibited a higher number of hypermethylated CpGs than the female-biased family in both ovaries and testes in control and treatment groups . The results showed a subset of them were significantly differentially methylated in different experimental groups in male-biased versus female-biased family of zebrafish (trmt10c and shmt2) and one chromatin-related gene (hmgn3) were significantly differentially methylated in four compared groups. Furthermore, some of the important reproduction-related genes  were identified in testes and ovaries of male-biased compared to female-biased family in both control and treatment groups. Of these, fmr1, which was also observed on the chromosome 14 in the MCMB vs. MCFB and MTMB vs. MTFB in Manhattan plots , male control female-biased versus female control female-biased (MCFB vs. FCFB), male treatment male-biased versus female treatment male-biased (MTMB vs. FTMB), male treatment female-biased versus female treatment female-biased (MTFB vs. FTFB) .The overall results showed a remarkable difference of DMCs in testes versus ovaries within both sex-biased families, where the DNA methylation landscape of the testes exhibited strikingly higher number of hypermethylated CpGs than the ovaries in both male-biased and female-biased families in control and treatment groups  for different positions associated with unique genes in each sex-biased family. The result indicated that the number of significant methylated unique genes was higher in male-biased (23 genes) than female-biased (6 genes) family in the control group. The same trend was observed in the treatment group, 28 genes in the male-biased and 14 genes in the female-biased family.To find the effect of DNA methylation changes on genes in male-biased and female-biased families, we annotated the DMCs with nearest genes comparing testes versus ovaries, resulting in the detection of unique genes (family-specific genes) in each family and common genes in overlap between the two sex-biased families . The resInvestigation of the overlap between candidate genes and the methylated unique genes in each sex-biased family  revealedFurther investigation of the overlap between candidate genes and common genes in the two sex-biased families, 8,954 genes in the control group  and 8,982 FC) illustrated the range of minimum to maximum value variation and the mean values in the compared groups corresponding to at least ten DMCs for each gene. Our findings showed that many of the reproduction-related genes  were highly methylated in the gonads of male-biased and female-biased families in both control and treatment conditions. Of these methylated genes, fmr1 and lztfl1 were associated with a strikingly high number of DMCs in all compared groups, over 288 genomic positions with fmr1 and 117 with lztfl1. Moreover, the methylation of some other reproduction-related genes, wt1a, prkcz, lhcgr, ints3, gsdf, was identified in the control group of both sex-biased families  in each family and common genes in overlap between the two sex-biased families . The resnabp1a and sox9b in the control group and histh1l and nabp1a in the treatment group of only male-biased family, where their methylated promoters were not detected in the female-biased family . The results of the functional analysis represent the biological function of the identified differentially methylated genes in the pathway and Gene Ontology (GO) categories. The important five GOs and pathways (p < 0.05) associated with the reproduction and epigenetic mechanisms in zebrafish gonad in the male-biased and female-biased families, based on the presented results in The annotated genes associated with DMCs in the comparison between the male-biased and female-biased families of zebrafish , 6 were The enriched GOs and pathways in the male-biased versus female-biased family differed markedly for both testes and ovaries as well as for control and treatment groups . ConsideThe enriched GOs and pathways in the testes versus ovaries revealed a considerable number of different GOs and pathways for each sex-biased family in the control and temperature groups . The resThe results of functional analysis of genes with differentially methylated promoters  in testeIn this study, we used zebrafish as a model animal to characterize sex ratio variation among different families and compared genome-wide DNA methylation in male-biased and female-biased families to gain new insights into the epigenetic regulatory mechanisms underlying sexual plasticity. Our findings on sex ratio variation among different families of this domesticated zebrafish strain support previous observations of the PSD system in domesticated zebrafish strains . In our A breeding experiment in a large number of domesticated zebrafish families revealed that broods derived from the same breeding pair exhibited similar sex ratio between repeated crosses, suggesting that sex in zebrafish is a heritable trait and parental genotypes have a strong influence on offspring sex ratio . This evThe expression of different phenotypes is encoded not only by the genetic information of the DNA sequence, but also by epigenetic modifications of chromatin structures including DNA methylation and alterations of histone proteins that bind DNA to regulate gene expression . The assEpigenetic marks can also be influenced by environmental factors . EpigeneTransgenerational inheritance of epigenetic modifications associated with differential DNA methylation regions (epimutations) is a plastic memory of organisms for phenotypic changes that can be transmitted to subsequent generations and cannot be explained by changes in primary DNA sequence and Mendelian genetics . Epigeneviml, mvb12bb, pitpnb, rtn1a, sp8b, olfml2ba, and fbxl22, located on chromosome 2, 8, 10, 13, 16, 20, and 25, respectively  within each sex-biased family comparing the testes and ovaries, where the male-biased family exhibited higher hypermethylated CpGs than the female-biased family, especially in the group exposed to high temperature . Therefofmr1 showed the highest number of associated DMCs in both families. This gene is involved in spermatogenesis and has a function in RNA regulation, which directly affects reproductive outcome by regulating the DNA damage response during spermatogenesis , which can be influenced by epigenetic regulatory mechanisms . In agreogenesis . Other imination . Consistnd lhx8a , was alsebrafish . Howeverogenesis . Howeversh gonad . Taken tcyp19a1a in fish species showed different methylation patterns during folliculogenesis in olive flounder, Paralichthys olivaceus (cyp19a1a promoter in European sea bass, Dicentrarchus labrax, results in suppression of its expression and male sexual development when incubated at high ambient temperature during early development (Oreochromis niloticus, also represented an increase in the methylation levels of different chromosomes in both sexes in elevated temperature-exposed fish compared to the control group (sox3 gene, resulting in up-regulation of its expression in both testes and ovaries (sox9a was increased only in the testes, but no significant difference was observed in the ovaries. Interestingly, the standard association between the hypermethylation and down-regulation of gene expression was not detected for sox9a in the testes at high temperature condition (Given that the DNA methylation of promoters as a functional regulatory element of the genome is highly related to gene expression , we purslivaceus  and zebrlivaceus . Likewiselopment . In addielopment . Global ol group . A recen ovaries . Howeverondition . Altogetondition , which mnabp1a, histh1l, and sox9b were methylated only in the male-biased family, that were not detected in the female-biased family (histh1l, a histone variant gene, was detected in the high incubation temperature group of the male-biased family, which may play a role in sexual development towards maleness. Consistent with our observation here, a previous study demonstrated the upregulation of histh1l in zebrafish testes compared to ovaries (Our further investigation of candidate genes comparing the two sex-biased families showed that the promoters of some reproduction- and epigenetic-related genes, including d family . Of thes ovaries .In summary, we found more DNA methylation in the male-biased family than the female-biased family and a high number of methylated positions in the testes than the ovaries. The effect of ambient temperature also showed more methylation at the high incubation temperature than the control temperature. These observations lead us to conclude that the DNA methylation changes may influence sexual phenotypic plasticity as a response to the environmental conditions. Our study provides new insights into the epigenetic mechanisms underlying sex-biased zebrafish families and improves our understanding of sex ratio variation in species with G × E mechanism of sex determination in evolutionary developmental biology.


There is limited research on associations between playing rugby union and player health post-retirement.This study investigated differences in self-reported sport injury history and current self-reported health characteristics between former New Zealand rugby and non-contact sport players with a view to identifying issues to be further investigated with stronger epidemiological research designs.NZ-RugbyHealth study surveyed 470 former rugby and non-contact sport players  recruited from October 2012 to April 2014. Demographic information, engagement in sport, sport injuries, medical conditions, mood, alcohol and substance use and ratings of current health status were obtained from a self-report 58-item general health e-questionnaire. We highlighted standardised differences in means of > 0.6 and differences in relative percentages of > 1.43 for variables between groups as representing at least moderate effect sizes, and of being worthy of follow-up studies.Using a cross-sectional design, the Higher percentages of the elite rugby player group had sustained injuries of a given body-site type  combination than the non-contact sports players. Higher percentages of the rugby groups reported having sustained concussion , injuries requiring hospitalisation , injuries that stopped participation in sport permanently  and sport-related surgery  during their playing career. Both rugby groups had a higher prevalence of osteoarthritis  than non-contact athletes and community rugby players had higher levels of hazardous alcohol consumption  in retirement than non-contact athletes. There was little difference between rugby players and non-contact sports athletes in self-reported mood, substance use and current physical or psychological health ratings.Former rugby player groups were at higher risk than the non-contact player group for most injuries during their playing careers, and in retirement had greater prevalence of osteoarthritis and hazardous alcohol consumption. The relative youth of the groups (43.8 years on average) means that health issues that typically do not emerge until later life may not have yet manifested.The online version contains supplementary material available at 10.1007/s40279-021-01630-7. NZ-RugbyHealth) project, containing four studies  were 1.1 [0.8–1.4] for elite rugby versus non-contact sport, 0.9 [0.6–1.1] for community rugby versus non-contact sport and 0.3 [0.1–0.5] for elite rugby versus community rugby. Analyses of self-rated health by sport group and self-reported concussions are provided in supplementary file 1, see electronic supplementary material [ESM]).Compared with the non-contact sport group, the two rugby groups reported substantially more concussions per player . Cohen's Current mood, medical conditions and health ratings of former player groups are presented in Table There were no substantial differences in self-reported mood between the player groups. A greater percentage of the elite rugby (38.2%) and community rugby player (40.3%) groups reported that they currently drink alcohol at hazardous levels (AUDIT Score > 8) compared with the non-contact sport (25%) player group. Mean scores on the AUDIT subscales were similar across the playing groups.NZ-RugbyHealth general health e-questionnaire cross-sectional study provided a snapshot of the playing and injury history, current demographics and health characteristics of former rugby and non-contact sport players and deliberately used several of the same research instruments as employed in RIPP. Our exploratory study formed an important step in helping to identify health issues amongst former rugby and non-contact sport players.The The hypothesis that rugby players are at substantially higher risk of injury than non-contact sport participants was supported by the results of our study. The elite rugby player group was at systematically higher risk of sustaining injuries during their playing careers than the non-contact sports player group; higher percentages of the elite rugby group were hospitalised because of a sporting injury and had a surgery related to an injury that occurred during participation in their sport than either the community rugby group or the non-contact sport player group. The proportions of the types of self-reported injuries were consistent with medically diagnosed injuries due to playing rugby from 2005 to 2017 recorded via the NZ national Accident Compensation Corporation claims [Injuries were also a greater factor in rugby players stopping playing their sport permanently; 28% of both the elite and community rugby groups stopped playing because of injury, compared with 11% of the non-contact sports players.UK-RugbyHealth study [UK-RugbyHealth study [Compared with the general population, former athletes have been found to have relatively high rates of osteoarthritis in the joints of the lower limb  and handth study  reportedth study . Sixty pth study , where tth study , where tNZ-RugbyHealth study were collected, the effects of concussive and sub-concussive head impacts on brain health have received increased governmental [Since the data for the rnmental , media [rnmental , 25 and rnmental –30. Arearnmental –33 and prnmental –37, moodrnmental –40, subsrnmental  and distrnmental .A systematic review of possible long-term effects of sports-related concussion in former athletes reported that multiple concussions appeared to be a risk factor for cognitive impairment and mental health problems in some individuals . DespiteNZ RugbyHealth cohort), it was concluded that there was “modest objective evidence” of decreased neuropsychological function in former rugby players. For ‘brain health’ the review included measures of cognitive function across eight domains . Therefore, it did not include information regarding specific diagnoses of neurological conditions such as Alzheimer’s or Parkinson’s disease . This is a gap in the literature that supports our recommendation for prospective longitudinal studies of brain health.In a 2018 review  of six pMany participants in rugby drink large quantities of alcohol as part of the cultural rituals of the sport . The misNew Zealand Medical Journal [Alcohol companies have a long history of sponsoring rugby union teams and competitions in New Zealand . Alcohol Journal  called f Journal  called f Journal . Multipl Journal . A numbe Journal . We acknTobacco companies have not been permitted to sponsor sports events in New Zealand since 1995. In the case of tobacco, government intervention to ban advertising at, and sponsorship of, sports events followed recognition of the deleterious effects of smoking tobacco products on the health of users. Use of tobacco among the retired players in our study (both rugby groups and non-contact athletes) was lower than for New Zealand males of a similar age (23%) . ConsideSubstance abuse can increase among people with traumatic brain injury . Given tA significant association between adverse alcohol use and osteoarthritis  has been reported in a group of former UK elite athletes   and beerData on health history including concussion were obtained from self-report, thus recall bias is a potential issue. The extent to which recall may be impaired, and whether any impairment differs systematically between groups, are important considerations when health histories are collected many years after exposure . FactorsWe sought to reduce potential inter-responder variability by asking whether conditions had been medically diagnosed; even so, self-reported health issues do not necessarily align with medically diagnosed health issues . Self-reLike all studies involving recruitment of volunteers, the study was subject to non-response bias. We originally planned to have 200 players in each group, but despite leaving the survey open for longer than originally intended and requesting (and obtaining) assistance for recruitment from NZ Players’ Associations, it was difficult at the time to recruit participants. The number of respondents in the elite rugby and non-contact groups were lower than intended, whereas we obtained more respondents than planned for the community rugby group.The non-contact sport group that was added to enable sports groups with and without collisions to be compared, had 26% who had sustained a concussion. As this group consisted of a mix of elite (43%) and community level non-contact sport players, there could have been regression to the mean within that group that may have affected the magnitude of the differences observed between groups.Non-response bias could have impacted the degree to which the samples are accurate representations of the wider population of players from which they were drawn. We were therefore conservative in interpreting the effects as representing important differences between the groups. Our interpretation of the standardised differences in means and the relative percentages highlighted in the tables is that there was a big enough difference between the groups to warrant follow-up with more robust research methods.Large inter-individual variation was common in the variables, which means we may be missing important real effects that hold in the wider population. Conversely, in some instances, we may be identifying effects that look important but are statistical artefacts produced through chance or the biases to which the study was prone. Due to low numbers we were not able to determine any differences in Alzheimer’s disease or dementia.Due to the cross-sectional nature of this study, causality between rugby/non-contact sport participation and demographics or general health in later life was unable to be determined. For example, we cannot eliminate the possibility that the former player groups may have differed in general health or demographic characteristics prior to participation in sport or from other aspects of their lives over their playing career. A strength of our study was that it enabled us to identify potential health issues associated with participation in rugby in a relatively short time span, to assist with planning of future follow-up studies. It remains unclear whether potential negative outcomes from injuries and alcohol use in rugby outweigh long-term health benefits associated with participation in physical activities per se. An interdisciplinary approach to the clinical care and support of former elite athletes after their careers has been advocated, as the interaction between physical and mental health issues occurring in the long term is complex .For athletes to be able to make informed decisions about the implications of playing rugby on their long-term health, they need quality health outcome and risk factor data. Longitudinal research on the health of rugby athletes over extended follow-up periods is needed to examine changes in health over time while accounting for demographic and health risk covariates, and to address non-response bias. The relative youth of the groups (43.8 years on average) means that health issues that typically do not emerge until later life may not have yet manifested.Former rugby player groups were at higher risk than the non-contact player group for most injuries during their playing careers. Following retirement, substantially higher percentages of the rugby player groups reported having been diagnosed with osteoarthritis and engaging in hazardous alcohol consumption than the non-contact player group. In terms of medical conditions, mood and life satisfaction, there were no substantial differences between former rugby players and non-contact sports people. All former player groups had lower rates of tobacco use than males of similar age in New Zealand.determining what the contributing factors to hazardous alcohol consumption are among rugby players/culture/environment;determining how players should be advised of the injuries they are likely to experience and the potential for longer term health issues related to these injuries, such as osteoarthritis, so they can make informed choices about engagement in rugby.Given the findings of the study, we recommend research to address implications for player general health in retirement from rugby, including:Supplementary file1 (PDF 426 KB)Below is the link to the electronic supplementary material.


Correction: Mol Neurodegener 13, 57 (2018)https://doi.org/10.1186/s13024-018-0286-0After publication of this work , an erroIn the Cell Tracker column shown below on the left, an incorrect representative image for Cell Tracker was used for the NI-IgG condition (non-immune IgG control). The error in this representative Cell Tracker image likely occurred when updates were made to the representative images in this figure during preparation of the manuscript by mistake. The Cy3-Aβ image for this NI-IgG condition was correct and does not need to be replaced.This error only pertains to the incorrect representative Cell Tracker NI-IgG image in Fig. After carefully going back through all the raw data, we found and selected the exact correct Cell Tracker image for this particular NI-IgG condition shown in Fig. We would appreciate to publish erratum and replace incorrect representative Cell Tracker image as shown on the left side below (as published) with the correct representative Cell Tracker image for this particular NI-IgG condition shown below on the right side.The correct version of the entire Fig.


The diagnosis of breast cancer with MRI is based on both morphological evaluation and kinetic curve assessment. Current computer-aided diagnosis methods for malignancy determination mainly focus on morphology features but ignored the temporal information in dynamic contrast-enhanced MRI sequences. Malignant and benign lesions usually have different enhancement patterns during the wash-in phase. Ultrafast breast MRI with high temporal resolution can capture the inflow of contrast in breast lesions. This advantage of ultrafast MRI enables the combination of both temporal and spatial information for automatic breast lesion analysis model development. We found that temporal information helps to significantly improve the performance of breast lesion classification. This suggests that ultrafast MRI provides useful information for malignancy identification and temporal information, which is indispensable for similar model development.Purpose: To investigate the feasibility of using deep learning methods to differentiate benign from malignant breast lesions in ultrafast MRI with both temporal and spatial information. Methods: A total of 173 single breasts of 122 women (151 examinations) with lesions above 5 mm were retrospectively included. A total of 109 out of 173 lesions were benign. Maximum intensity projection (MIP) images were generated from each of the 14 contrast-enhanced T1-weighted acquisitions in the ultrafast MRI scan. A 2D convolutional neural network (CNN) and a long short-term memory (LSTM) network were employed to extract morphological and temporal features, respectively. The 2D CNN model was trained with the MIPs from the last four acquisitions to ensure the visibility of the lesions, while the LSTM model took MIPs of an entire scan as input. The performance of each model and their combination were evaluated with 100-times repeated stratified four-fold cross-validation. Those models were then compared with models developed with standard DCE-MRI which followed the same data split. Results: In the differentiation between benign and malignant lesions, the ultrafast MRI-based 2D CNN achieved a mean AUC of 0.81 ± 0.06, and the LSTM network achieved a mean AUC of 0.78 ± 0.07; their combination showed a mean AUC of 0.83 ± 0.06 in the cross-validation. The mean AUC values were significantly higher for ultrafast MRI-based models than standard DCE-MRI-based models. Conclusion: Deep learning models developed with ultrafast breast MRI achieved higher performances than standard DCE-MRI for malignancy discrimination. The improved AUC values of the combined models indicate an added value of temporal information extracted by the LSTM model in breast lesion characterization. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) has a higher sensitivity compared with other modalities for breast cancer detection . It is wDuring DCE-MRI scanning, the concentration of gadolinium contrast material in the permeable blood vessels of tumor tissue causes a decrease in the relaxation time of water protons and results in a high signal in T1-weighted images. The current DCE-MRI protocol usually contains multiple T1-weighted acquisitions after contrast. The diagnosis of breast lesions is highly dependent on the high spatial resolution and kinetic pattern in the wash-out phase, making MRI scanning tedious. To enable a wider use of breast MRI, protocols should be shorter to improve cost effectiveness ,5. DiffeMost abbreviated protocols are based on T1-weighted imaging before and after contrast agent administration. T1-weighted acquisitions may be performed in different time resolutions. Conventional T1-weighted MRI has high spatial resolution, but is time consuming, while the time-resolved angiography with stochastic trajectories (TWIST) technique enables ultrafast acquisition at the cost of a lower spatial resolution ,15. CompRecently, artificial intelligence (AI) was introduced to help discriminate benign from malignant lesions in breast MRI ,21. MostWe hypothesized that fully automatic deep learning-based methods, with only ultrafast DCE-MRI as input, could be used for malignancy identification. Therefore, the aim of this study is to evaluate the feasibility of this automatic deep learning-based method and investigate the usefulness of temporal information extracted with an LSTM model.This retrospective study was approved by the local Medical Ethics Review Board (METc Nr: 2018/652) with a waiver of the requirement for informed consent from each patient. A total of 1447 breast MRIs, including both screening and diagnostic examinations, from 809 patients who underwent breast MRI scans at our hospital between 2016 and 2019 were retrospectively included. Among the 1447 consecutive breast MRIs, 443 were excluded because no ultrafast DCE-MRI sequences had been acquired. Furthermore, 694 MRI scans from 374 patients with only focus/foci  or with no lesion reported were excluded. A total of 34 MRI scans for chemotherapy evaluation, 31 for breast implants, and 94 post-operative scans were also excluded, resulting in 151 MRI scans from 122 patients. For the remaining 151 MRI scans, left and right breasts were assessed separately for the presence of breast lesions. A total of 129 breasts with no suspicious lesion were excluded, and 173 breasts with one or more lesions were included in the final model development. The label of each breast was determined by the primary lesion contained. For breasts with no malignant lesions, the benign lesion with the biggest size was analyzed. For breasts with malignant lesions, the possible additional present benign lesions were not included in the analysis. The ground truth of those included lesions was obtained from pathology reports (surgery or core needle biopsy) or follow-up examinations . The final study dataset contained 109 benign and 64 malignant lesions .All DCE-MRI scans were acquired on a 3 T  or 1.5 T  scanner with a circularly polarized bilateral breast coil (Siemens). The protocol consisted of a pre-contrast T1 and T2 and the following sequences after contrast: DWI, TWIST, and four DCE-T1-weighted series. A measurement of 15 mL of contrast agent  was injected using a power injector. Acquisition parameters of the T1-weighted and TWIST sequence are list in Breast segmentations were first performed by using a 3D U-Net  to removTo take advantage of both morphological and temporal information provided by ultrafast breast MRI, a CNN model and a long short-term memory LSTM network were employed to extract morphological and temporal features, respectively.  The CNN The output of each model was a numerical value between zero and one, which indicates the probability of malignancy. The performance of each model and their combination was evaluated with 100-times repeated stratified 4-fold cross-validation. During inference, the CNN models assessed each of the four MIP images from a single validation sample, and the highest output value was then determined as the malignancy probability of the sample. The output of the proposed system was then determined by averaging the predicted scores from both models. The receiver operating characteristic (ROC) curve was plotted for each model and the entire system based on the predicted probability. Sensitivity and specificity were used to evaluate the performance of each model with different thresholds applied.Models were developed with Pytorch framework (v1.9.1) and trained with an NVIDIA Quadro P4000 GPU. For the 2D CNN (ResNet-18) model, transfer learning was adopted with pre-trained weights on ImageNet; convolution layers were frozen while only fully connected layers’ parameters were finetuned. The training was run for 30 epochs with a learning rate of 1e-4, a batch size of 16, and a momentum of 0.9. The Adam optimizer and a categorical cross-entropy loss function were used. For the LSTM model, a ResNet-18 model was used as feature extractor. The same hypermeters were used for training, except the batch size was reduced to four and training epochs were extended to 40. During training, MIP images were first resized to 224 × 224, and then sent into the models; random horizontal flip (probability of 0.5) and random rotation , but the combined prediction could further improve the performance . There is clearly an added value in using the temporal information extracted by the LSTM model. A detailed analysis of the diagnosis performance with different cutoff values is listed in The 2D CNN achieved a higher mean AUC than LSTM (To better illustrate the classification patten of the models developed with ultrafast MRI, boxplots of the mean AUC values of each model during cross-validation are illustrated in In this study, we investigated the possibility of using a deep learning model for breast lesion discrimination with only ultrafast MRI as input. Motivated by recently discovered kinetic features derived from ultrafast MRI, we looked at the effectiveness of deep learning-based automatic pharmacokinetic analysis of the wash-in phase and possible improvements to morphological analysis. For this purpose, a 2D CNN model and an LSTM model were first trained and evaluated separately for the classification of benign and malignant lesions, and then combined by the fusion of the predicted scores of each model. The results showed that deep learning models could achieve high performance for lesion differentiation with only ultrafast breast MRI. The models developed with ultrafast MRI had a significantly higher mean AUC values compared with the model developed with standard DCE-MRI. Meanwhile, the combined model achieved the highest mean AUC value during repeated straited cross-validation. Even though the mean AUC value of the LSTM model was lower than the 2D CNN model, after integrating them together, the temporal information extracted by the LSTM model turned out to have added value for diagnostic performance. This upward trend was observed with both ultrafast and standard MRI.Machine learning was also adopted for malignancy prediction in previous studies with different sequences. Platel et al.  used 5 mCompared with previous studies developing classifiers with handcraft or radiomics features, in this work, we used a 2D CNN model and an LSTM model to automatically extract morphological and kinetic features separately. Instead of highly depending on the manually delineated lesion area for feature extraction, a big advantage of the proposed method is that neither manual annotation nor dedicated segmentation of the lesions are required before analysis. The models take raw images as input and provide predictions based on the content only; therefore, it could be easily integrated in the clinic workflow and works fully automated. What should also be noticed is that there is no associate relationship of identification ability between machine learning models and radiologists. Radiologists still rely more on high-resolution T1-weighted sequences for malignancy discrimination, which intuitively contradicts the finds of this paper. A possible explanation for the inferior performance of the standard DCE-MRI-based models might be that temporal analysis of the TWIST sequence with the LSTM model made up for the defect of morphological analysis caused by insufficient spatial resolution.Despite these promising results achieved with ultrafast MRI sequences, we can easily envision the improvements when taking other sequences into consideration. For accurate diagnosis, delayed sequences are indispensable in clinical practice, especially the wash-out phase in DCE-MRI which provides irreplaceable functionality of indicating malignancy. However, in the trend of shortening MRI protocol for screening, identifying the malignancy of a tumor from an easily accessible sequence with a fully automatic model could provide support for a flexible scanning strategy, in which additional MRI sequences could be added from the protocol as needed. The flexibility of MRI could save time while maintaining the performance of breast cancer screening.There are still several limitations of our study. This retrospective study had a relatively small dataset from a single medical center, and not all the benign lesions were pathologically confirmed. Even though the dataset has been randomly split 100 times to illustrate the robustness of the models, a large multivendor multicenter dataset is still needed for further validation. Another limitation of the work is that the study was conducted in a high-risk screening population and included pre-operation examinations; therefore, the prevalence of malignant lesions in our study is higher compared with the general screening population, which may weaken the model’s generalizability. Additionally, patient information has been proven effective to improve the decision-making of classification models, and properly integrating patient information may further improve the performance. Furthermore, we focused on the validation of the usefulness of temporal information extracted with an LSTM model; all the models were trained with the same hyperparameter setting and training strategy, which may lead to an underestimated performance of the system. Finally, models were trained and validated with the output of the primary lesion in each breast. Therefore, the predictions only revealed the status of primary lesions but overlooked associated lesions. Object detection models, which could locate each single lesion, may help address this problem.In conclusion, deep learning models achieved promising results for the fully automatic differentiation of benign and malignant lesions. Kinetic information extracted by LSTM model has added value in the 2D CNN-based morphology analysis.


Point-of-care ultrasound (POCUS) plays a prominent role in the timely recognition and management of multiple medical, surgical, and obstetric conditions. A POCUS training program for primary healthcare providers in rural Kenya was developed in 2013. A significant challenge to this program is the acquisition of reasonably priced ultrasound machines with adequate image quality and the ability to transmit images for remote review. The goal of this study is to compare the utility of a smartphone-connected, hand-held ultrasound with a traditional ultrasound device for image acquisition and interpretation by trained healthcare providers in Kenya.This study took place during a routine re-training and testing session for healthcare providers who had already received POCUS training. The testing session involved a locally validated Observed Structured Clinical Exam (OSCE) that assessed trainees’ skills in performing the Extended Focused Assessment with Sonography for Trauma (E-FAST) and focused obstetric exams. Each trainee performed the OSCE twice, once using a smartphone-connected hand-held ultrasound and once using their notebook ultrasound model.Five trainees obtained a total of 120 images and were scored on image quality and interpretation. Overall E-FAST imaging quality scores were significantly higher for the notebook ultrasound compared to the hand-held ultrasound but there was no significant difference in image interpretation. Overall focused obstetric image quality and image interpretation scores were the same for both ultrasound systems. When separated into individual E-FAST and focused obstetric views, there were no statistically significant differences in the image quality or image interpretation scores between the two ultrasound systems. Images obtained using the hand-held ultrasound were uploaded to the associated cloud storage using a local 3G-cell phone network. Upload times were 2–3 min.Among POCUS trainees in rural Kenya, the hand-held ultrasound was found to be non-inferior to the traditional notebook ultrasound for focused obstetric image quality, focused obstetric image interpretation, and E-FAST image interpretation. However, hand-held ultrasound use was found to be inferior for E-FAST image quality. These differences were not observed when evaluating each E-FAST and focused obstetric views separately. The hand-held ultrasound allowed for rapid image transmission for remote review.The online version contains supplementary material available at 10.1186/s13089-023-00308-7. The use of point-of-care ultrasound (POCUS) benefits patient screening, the accuracy of diagnosis, and management for a wide range of indications in low- and middle-income countries (LMICs) , 2. SeveIn 2013, we developed a program to train rural healthcare providers in Kenya on POCUS applications including the Extended Focused Assessment with Sonography for Trauma (E-FAST) and focused obstetric ultrasonography . The proDue to geographic isolation, trainees lack frequent real-time guidance and feedback while scanning at their facilities and have limited interactions with trainers outside of the scheduled sessions 3 times a year. Workforce limitations mean that many trainees are unable to arrange time away from their healthcare facilities to attend training in the capital city. The current ultrasound system that was donated for their use does not allow for image transmission for remote review and quality assurance. Poor infrastructure and limited Internet connectivity further prevent the creation of robust frameworks for image review and feedback .The cost of ultrasound machines has historically prohibited the expansion of POCUS use in LMICs. However, this challenge is more surmountable with the development of more cost-effective hand-held ultrasound models. These ultrasounds present a significantly more affordable option for full-body imaging in low-resource areas. They also improve portability for bedside use. Importantly, these ultrasound models incorporate cloud-based storage systems where images can be immediately uploaded for expert review and feedback.Several studies have evaluated the efficacy of hand-held ultrasounds and acknowledged their potential benefits –14. One The primary aim of this study was to introduce a hand-held ultrasound model into our training program in rural Kenya and to compare image quality and image interpretation by POCUS trainees using the hand-held device compared to the notebook ultrasound model in current use. We also sought to determine the median time required to transmit ultrasound images obtained using the smartphone-connected hand-held ultrasound model through local cell phone networks.This was a pilot study designed as a non-inferiority test of the hand-held ultrasound compared to a notebook ultrasound model currently in use by the Kenyan POCUS trainees. We recruited healthcare providers who had received prior POCUS training and were presenting for follow-up refresher training and testing. All participants were above 18 years of age and provided consent to participate. The study took place in April 2018.The refresher session entails a full hands-on review of all the point-of-care ultrasound modalities that the trainees have been exposed to before. I.e., The E-FAST and focused first- and second/third-trimester obstetric ultrasound. These exams are carried out on healthy, pre-scanned volunteers who provide informed consent. At the end of the refresher program, trainees are evaluated on their ultrasound scanning skills using a Standardized Observed Structured Clinical Exam (OSCE)  that was connected to an iPhone 8. The trainees practiced using this ultrasound model to perform guided E-FAST and focused obstetric exams. Afterward, we performed the OSCE evaluation twice for each trainee: once using the current notebook ultrasound model  and once using the Butterfly iQ probe. The OSCE testing was carried out on a separate group of pre-scanned volunteers. The order of ultrasound use and ultrasound modality testing was randomized to minimize bias from the previous examination. This was achieved by creating a randomized order in which the trainees rotated through different testing stations. Each testing station had a different volunteer/model, half the stations were equipped with a hand-held ultrasound, and the other half were equipped with a notebook ultrasound.Images were scored independently by three investigators who are credentialed in POCUS according to the American College of Emergency Physician guidelines. Two of these investigators had also completed fellowships in acute and critical care ultrasound. A standardized scoring system was used to grade image quality on a scale of 0 to 4 . This scoring system is currently in use in the Kenya rural ultrasound training program  as well Image interpretation was scored using a standardized scoring system from 0 to 3:   reported. Wilcoxon tests were conducted to determine differences between the notebook and hand-held imaging systems on overall E-FAST and focused obstetric image quality and image interpretation. A series of Wilcoxon tests were also performed to determine differences between the two ultrasound systems on each separate E-FAST and focused obstetric view, e.g., identifying differences in image quality and image interpretation for Right Upper Quadrant (RUQ) and Left Upper Quadrant (LUQ) views separately. Within each view category (E-FAST and obstetric), the scores for the two internal reviewers were averaged. The score of the external reviewer was used as a validity check for the internal consistency of the two primary internal reviewers. Agreement rates between the two internal reviewers and the external reviewer were calculated for image quality. Agreement within 1 point between the internal and external reviewers was considered as agreement.The study was approved by the Aga Khan University Research Ethics Committee (REC) and the National Commission for Science, Technology, and Innovation (NACOSTI) Kenya.Data were collected from five trainees who conducted a series of ultrasound imaging using the hand-held and notebook ultrasound. Each trainee obtained six E-FAST views and six focused obstetric views on each ultrasound machine, a total of 24 views for each trainee, resulting in 120 total ultrasound images assessed in this study. Image quality was scored on a 0–4 point scale and image interpretation was scored on a 0–3 point scale by three trained raters. Where a trainee obtained multiple images or clips in each view , the highest score per view was documented.p = 0.04]. The overall median interpretation scores were 3  for the notebook ultrasound and 3  for the hand-held ultrasound [p = 0.67]. For obstetric views, overall median image quality scores were 3.5  for the notebook ultrasound and 3  for the hand-held ultrasound [p = 0.18]. The overall median interpretation scores were 3  for the notebook ultrasound and 3  for the hand-held ultrasound [p = 0.06].For E-FAST views, the overall median image quality score was 3.5  for the notebook ultrasound and 3  for the hand-held ultrasound [Agreement between the internal and external raters reached 53% for E-FAST image quality for the notebook ultrasound and 57% for the hand-held ultrasound. For obstetric views, agreement on image quality was 80% for the notebook and 83% for the hand-held ultrasound.A series of Wilcoxon tests were conducted on differences in image quality and image interpretation by ultrasound system for the specific E-FAST and obstetric views as shown in Table Images and short clips obtained using the smartphone-connected hand-held ultrasound were uploaded to the associated cloud storage using a local 3G-cell phone network. Upload times were 2–3 min.After a 2-h introduction to Kenyan rural POCUS trainees, we found the hand-held ultrasound to be non-inferior to their notebook ultrasound on overall measures of E-FAST image interpretation, focused obstetric image quality, and interpretation. However, the hand-held ultrasound was found to be inferior in overall measures of E-FAST image quality. When comparing separate imaging views, no statistically significant difference was found between the notebook ultrasound compared to the hand-held ultrasound.Our study findings are comparable with results from a single-center prospective observational study with 307 participants in Sierra Leone where a hand-held ultrasound device was found to be as reliable as the traditional ultrasound device in structured obstetric ultrasound exams . The SieDespite our findings showing the non-inferiority of the smartphone-connected hand-held ultrasound, trainees brought up several challenges such as difficulty with the iPhone screen size, especially with the need to use a split screen to calculate fetal heart rate. Second, trainees were challenged by the lack of built-in fetal biometric parameters which were not available at the time of this study. It is possible that optimization of the obstetric modality coupled with the ability to use the hand-held probe connected to a larger screen might further improve image quality and interpretation for obstetrics.Our trainees underperformed on E-FAST image quality scores when using the hand-held ultrasound. The short 2-h introduction to the hand-held ultrasound may have played a part in this. In addition, our previous study in the same population of trainees showed that they do not perform the E-FAST as often as they perform focused obstetrics . HoweverThere are notable limitations in this study. The sample size of 120 images obtained by 5 trainees was small. The small sample size was further reduced when we separated and evaluated specific ultrasound views, e.g., RUQ and LUQ. Though the study size is small, it has the benefit of comparing images acquired by multiple novice POCUS trainees. Other studies comparing the efficacy of hand-held and traditional ultrasound devices have relied on comparing the images among a single experienced ultrasonographer , 12. TheFinally, the percent agreement between the internal and external rater was good for obstetric image quality (80% for the notebook ultrasound and 83% for the hand-held ultrasound) but poor for E-FAST image quality (53% for the notebook ultrasound and 57% for the hand-held ultrasound). This affects our interpretation of the E-FAST image quality results. The agreement in obstetric image quality results is comparable to the study by Kodaira et al. in Sierra Leone . In studThis study found that it took 2–3 min to upload ultrasound images from the hand-held device to a secure cloud storage platform. Image upload and transmission were achieved via a local 3G cell phone network. Hand-held devices that allow for rapid image transmission through local cell phone networks have great potential to expand POCUS training through remote image review and feedback .In this non-inferiority trial, the use of a hand-held ultrasound was found to be non-inferior to a traditional notebook model for focused obstetric image quality, obstetric image interpretation, and E-FAST image interpretation, and found to be inferior to the notebook model for E-FAST image quality. When segregated into different ultrasound views, the hand-held ultrasound was found to be non-inferior for each of the views evaluated. In addition, the hand-held device allowed for rapid image transmission via local cell phone networks. This is highly desirable for POCUS training programs in low-resource settings seeking to establish frameworks for remote image review and feedback. Future studies could test the performance of hand-held ultrasounds which now have improved specifications among a larger group of trainees. Further, studies that include clinical implications will provide useful data on the impact of hand-held ultrasound use on patient outcomes. We believe that this will inform current efforts to improve access to POCUS training in low-resource settings.Additional file 1. Appendix A: Observed Structured Clinical Exam Assessment (OSCE) Form.


Epigenetic modifications of histones  are of great importance in determining the functional state of chromatin. Changes in epigenome underlay all basic biological processes, such as cell division, differentiation, aging, and cancerous transformation. Post-translational histone modifications are mainly studied by immunoprecipitation with high-throughput sequencing (ChIP-Seq). It enables an accurate profiling of target modifications along the genome, but suffers from the high cost of analysis and the inability to work with living cells. Fluorescence microscopy represents an attractive complementary approach to characterize epigenetics. It can be applied to both live and fixed cells, easily compatible with high-throughput screening, and provide access to rich spatial information down to the single cell level. In this review, we discuss various fluorescent probes for histone modification detection. Various types of live-cell imaging epigenetic sensors suitable for conventional as well as super-resolution fluorescence microscopy are described. We also focus on problems and future perspectives in the development of fluorescent probes for epigenetics. Epigenetic modifications provide genome plasticity and enable cells to express a specific set of genes. They include DNA methylation, diverse modifications of histone proteins and also the modulation of gene expression in cis by coating DNA with non-coding RNA . Histones are basic proteins that form a structural scaffold in chromatin and undergo a significant degree of post-translational modifications mainly in their N-terminal tails. These changes serve as recognition sites for a variety of chromatin-binding proteins, as well as regulating steric access to DNA for replication and transcription machinery. Understanding histone modification dynamics is thus critical for deciphering the mechanisms of epigenetic regulation.One of the major mechanisms for this regulation is post-translational modifications—methylation, acetylation, phosphorylation, and some others—that are abundant mainly at the N-terminal regions of histone proteins, especially histones H3 and H4. There are protein domains that are responsible for introducing modifications to histone molecules (“writer” enzymes) and removing these modifications (“eraser” enzymes). In addition to changing the physicochemical properties of histones , modificDespite their critical roles in regulating chromatin architecture, it is unknown how epigenetic modifications are dynamically coordinated with chromatin remodeling. Therefore, the development of sensitive and precise methods for epigenome tracking, ideally within intact living cells is critical. Monoclonal antibodies that specifically bind with various post-translational modifications of histones play an important methodological role in the study of this problem . Such anAnother way to characterize individual cells can be fluorescence microscopy, due to its relative simplicity and the rich spatial information provided. In this review, we discuss fluorescence imaging strategies for detecting histone modifications in both fixed and living samples.Immunofluorescence is a classical approach to image various epitopes, including modified histones, using specific antibodies . In thisIn 2019, Terskikh and coworkers proposed a new method called Microscopic Imaging of Epigenetic Landscapes (MIEL) . At the Terskikh and co-authors have shown that MIEL allows: (1) to identify epigenetic landscapes characteristic of various cell lines; (2) to detect changes in chromatin under the influence of various drugs, while drugs of the same mechanism of action lead to similar changes in the landscapes of histone modifications; (3) monitor chromatin changes during cell differentiation . High-throughput sequencing (RNA-Seq) confirmed a high degree of correlation between MIEL cell comparisons and standard transcriptome analysis. Thus, MIEL is a promising tool for revealing differences in the epigenetic state of cells in various biological models, as well as for the high-throughput screening of drugs .In 2020, Hayashi-Takanaka et al. described the method for providing detailed information on histone modifications using multicolor immunofluorescence-based single cell analysis . They foRecently, Takei et al. developed the new approach to characterize nuclear architecture using a combination of DNA seqFISH+, multiplexed IF and RNA seqFISH methods . The groAnother straightforward approach was published in February 2022—Rong Fan group developed the method that they called Spatial-CUT&Tag . It explHowever, immunofluorescent analysis of chromatin is limited to fixed samples only. It cannot be used to track the dynamics of epigenetic changes in live cells, for example, in the course of cell differentiation or drug treatment. To fill this gap, a number of genetically encoded epigenetic probes were developed as discussed below.Antibody immunostaining is currently the best and unique method for imaging proteins and different targets, and the potential use of antibodies for live-cell imaging is an ideal option. Histone modifications are good targets for synthesized antibodies, but whole antibodies cannot be used within living cells. Immunoglobulin G (IgG), used for immunostaining, consists of four chains: two light (L) and two heavy (H). These chains form the constant (Fc) and antigen-binding (Fab) region, which forms the complementary-determining region (CDR). Because IgG’s size of 150 kDa prevents it from passing through nuclear pores, reduced variants of antibodies have been developed. The monovalent antibody fragment (Fab) has a size of 50 kDa and has the ability to pass through nuclear pores [The first approach for monitoring the distribution of histone modification in live cells, based on fluorescently labeled Fab fragments, was invented in 2011 and called FabLEM . PrimariAdditionally, Fab fragments were used in the study of H3S10 histone modification in living cells , the effThe development of genetically encoded probes based on antibodies was the next step in the live-cell imaging of post-transcriptional modifications of histones. Yuko Sato et al.  created Additionally, H3K9ac mintbody was used in Xenopus laevis  and in pAs mentioned earlier, scFvs are truncated variants of full-length antibodies that only contain the variable domains of heavy (VH) and light chains (VL) . Camelid2+ [Basically, genetically encoded Förster Resonance Energy Transfer (FRET) sensors consist of two fluorescent proteins flanking a recognition domain and forming a single polypeptide chain A. Ligand2+ . After t2+ . Along w2+ ,30. smFR2+ , transcr2+ , structu2+ , etc. sm2+  and the 2+ .The schematic representation of FRET-based epigenetic sensors described below is presented in The first FRET-based sensor specific for acetylation was published by Kanno et al. . The resPeng et al. created a FRET biosensor to visualize the histone H3 Lys-9 trimethylation (H3K9me3) dynamics in single live cells . When H3Recently, Han et al. proposed a new variation of the sensor for histone H4 acetylation state detection . The autd 1–200 μM for chromodomains and 3–300 μM for bromodomains). Using the intrabody as a sensor and a FRET fluorescent–protein pair as reporters, the authors took advantages of both methods and could monitor histone-modification levels by ratiometric FRET quantification in living cells. The authors showed that the lack of the H3 tail in the probe had no significant effect on FRET response, indicating that the probe binds to endogenous acetylated histone H3K9 rather than the H3 sequence within the probe to increase its FRET efficiency [Chung et al. proposed to use an intrabody-based FRET probe, which represents the combination of FRET methodology with previously described acetyl H3K9-specific scFv . The auficiency . Anotherficiency . They inDespite its certain advantages, FRET experiments are quite challenging to conduct due to the fact that FRET is dependent on many factors, such as close proximity, large amounts and certain stoichiometric ratios of fusion proteins in order to obtain valid data. The process of measuring and quantifying the FRET results is also difficult .Another valuable method for imaging the protein–protein interactions in general and epigenetic landscape dynamics specifically is bimolecular fluorescence complementation (BiFC). The method itself is based on splitting a single fluorescent protein into two parts and attaching the parts to the proteins thought to interact within cells. The split fragments themselves lack fluorescence unless they are brought in close enough proximity to become activated .A good example of the method application is the work by Vincenz and Kerppola who utilized the BiFC assay to look into the role of the conserved regions of chromatin-binding CBX proteins in their recruitment to certain chromatin regions alongside with the significance of the H3K27me3 modification in this process .CBX is a part of the polycomb repressive complex 1 (PRC1) and has the ability to bind H3K27me3 . It was To test this theory, several probes were produced containing each of the CBX family members and the first fluorescent protein fragment. The second part of the fluorescent protein was fused to the histone H3 isoforms. Strikingly, the results provided by the quantitative BiFC assay showed that CBX recruitment to the chromatin regions was mediated strictly by non-conserved domains of the proteins. Interestingly, the PRC1 recruitment dependence on the PRC2 activity was not confirmed. It was clearly shown that H3K27 trimethylation was not required for CBX-chromatin interaction in live cells. Moreover, chromatin-bound CBX proteins did not exhibit colocalization with the H3K27me3 modification outside the inactive X.In a more recent study, a similar approach was used by Sekar et al. . The autIn the designed sensors that activate themselves upon the methylation of either H3K9 or H3K27, specific substrate domains derived from H3 histone proteins are accompanied by chromodomains either from Suv39H1 protein or from HP1 protein. These units are inserted between the N- and C-terminal domain of the split-Renilla luciferase protein (RLuc8.6). Upon the binding of the sensor to the methylated histone tails, the optimal complementation of the luciferase is achieved, thus reconstructing its enzymatic activity and activating its bioluminescence properties. The efficacy of the sensor was evaluated in the experiment where cells stably expressing the probe were treated with methyltransferase inhibitors (Bix01294 and UNC0638). According to the obtained results, a concentration-dependent decrease in luciferase bioluminescence was observed.Another interesting trait of this exact research was the successful usage of the novel sensor in vivo. The authors were able to assess the histone methylation landscape in mice carrying tumor xenografts derived from the sensor-expressing tumor cells. They further tested the sensor in response to the intratumoral injection of Bix01294 and, as expected, observed a significant drop in luciferase complementation levels. Such promising results make this sensor a good candidate for preclinical utilization.Another group of researchers focused their efforts on the direct detection of changes in pericentromeric H3K9me3 levels and studied the dynamics of these changes upon drug treatment . CentromIn a very recent work, Ohmuro-Matsuyama et al. addressed the issue of visualization of histone acetylation marks and the effects of histone deacetylase (HDAC) inhibitors . They foCaenorhabditis elegans [C. elegans is essential for bacterial aversion behavior itself as well as for the transmission of such kind of behavior onto the next generation, but also that PAR-5 protein (a member of 14-3-3 chaperone protein family) is an important H4K8ac-interacting partner and may be involved in its regulation. That was achieved by engineering the BiFC constructs with fragments of GFP (green fluorescent protein) fused with PAR-5 and H4. The occurrence of the interaction between the two proteins of interest was confirmed with the fluorescence observed in vivo.Another interesting work, also issued this year, tackled the issue of gut-governed behaviors in  elegans . The resDespite their numerous advantages, the BiFC method has some serious shortcomings that are to be considered. The most important one is that BiFC formation is irreversible. This can be useful in detecting weak interactions, but makes them useless in studying dynamic protein complexes . On the Histone modification reader domains (HMRD) are key players in the interpretation of the histone code by binding to modified histones and attracting proteins with specialized functions required at a given chromatin locus . Reader Firstly, a considerable number of scientific groups reported the crystal structures of reader domains, described their binding properties and determined their dissociation constants: for hMPP8 (MPHOSPH8) ,70, ATRXThen, Kungulovski et al. performed a proof-of-principle study, in which they characterized the specificity of several engineered histone modification-interacting domains (HMIDs) using the CelluSpots histone peptide arrays and compared them with ENCODE-validated antibodies for the same PTM . It was After that, the idea that recombinant HMRDs can be used to create genetically encoded fluorescent sensors to monitor the activity of histone-modifying proteins in living cells was implemented. The same group, which previously described HMIDs, published a variant of fluorescent sensor with double specificity: it consisted of two fused recombinant histone modification-interacting domains (HiMIDs) for the direct detection of loci with doubly modified chromatin . The autIn 2018, Delachat et al. developed chromatin-sensing multivalent probes (cMAPs) by combining two HMRDs with different specificities in one construct to visualize loci in which two different modifications coexist . The autSanchez et al. also created the heterodimeric sensor visualizing H3K9me3 modification in living cells  Figure B. The auIn 2020, Villaseñor et al. developed ChromID—a method for identifying the chromatin-dependent protein interactome . The autSummarizing, the reader domain-based workflow can be a powerful tool for our deep understanding of the functioning of distinct chromatin states and reader domains in gene regulatory mechanisms.Our current understanding of higher-order chromatin structures defined by distinct histone modifications is derived indirectly from in vitro biochemical experiments, such as chromatin immunoprecipitation (ChIP), due to the poor resolution of traditional light microscopy ,83 and cIn the work by Conic et al., the authors were successful in high-resolution live-cell imaging. They offered the electroporation step for intracytoplasmic delivery labeled primary antibodies and Fabs into cultured cells and could image the dynamics of phosphorylated histone H2AX . The autOther groups went further and established a goal to describe the detailed information of epigenetic modifications important for cellular functions and activities lacking in previous experimental designs. Xu, Liu et al.  used locPrakash et al. aimed to demonstrate the power of SMLM to describe the epigenetic landscape of pachytene chromosomes . They reIn general, observing histone modifications at the single-molecule level can contribute to describe the roles of epigenetic modifications in higher-order genomic architecture.Recent years have been characterized by a high interest in research at the level of single cells . For exaSeveral methods mentioned above, such as MIEL , HiMIDs The current implementation of MIEL and HMRD-based methods is limited by the light diffraction barrier: all fluorescent spots located closer than ~200 nm are indistinguishable from one another. It may be useful to extract more information from the epigenetic landscape using super-resolution fluorescence microscopy . MoreoveAnother limitation of data obtained through the live imaging of histone marks is the lack of correspondence between the epigenetic landscape and the cell cycle phase in an individual cell. To gain such information, it may be useful to implement Fluorescent Ubiquitin-based Cell Cycle Indicator (FUCCI) ,104.A further promising direction is the application of phototoxic fluorescent proteins , such asThe most challenging task is to correlate intranuclear fluorescent patterns with sequence-based profiles of corresponding epigenetic modifications. Data for selected specific loci can be obtained by their co-labeling using established techniques such as fluorescence in situ hybridization (for fixed cells) or endonuclease-deficient Cas fused with fluorescent tag (for live or fixed cells) . A large


Efficient scanning of tissue that T cells encounter during their migratory life is pivotal to protective adaptive immunity. In fact, T cells can detect even a single antigenic peptide/MHC complex (pMHC) among thousands of structurally similar yet non-stimulatory endogenous pMHCs on the surface of antigen-presenting cells (APCs) or target cells. Of note, the glycocalyx of target cells, being composed of proteoglycans and bulky proteins, is bound to affect and even modulate antigen recognition by posing as a physical barrier. T cell-resident microvilli are actin-rich membrane protrusions that puncture through such barriers and thereby actively place the considerably smaller T-cell antigen receptors (TCRs) in close enough proximity to APC-presented pMHCs so that productive interactions may occur efficiently yet under force. We here review our current understanding of how the plasticity of T-cell microvilli and physicochemical properties of the glycocalyx may affect early events in T-cell activation. We assess insights gained from studies on T-cell plasma membrane ultrastructure and provide an update on current efforts to integrate biophysical aspects such as the amplitude and directionality of TCR-imposed mechanical forces and the distribution and lateral mobility of plasma membrane-resident signaling molecules into a more comprehensive view on sensitized T-cell antigen recognition. Slight deviations within the involved mechanisms can lead to severe medical complications, namely, allergies, autoimmune diseases, hypo- or hyper-reactions to invading pathogens, and the development of cancer. The degree of fine-tuning required for immune protection becomes apparent at multiple regulatory levels controlling T-cell activation. The pivotal event preceding many of the ensuing cellular interactions concerns the specific molecular recognition of processed pathogenic peptides displayed in the context of the major histocompatibility complex  by T cells sine qua non considering the consequences of recognition failure. It is, however, not yet clear how such a level of specificity and sensitivity is maintained, since measured biochemical affinities between TCRs and pMHCs are astonishingly low. A number of models have been conceived and tested in the last two decades. Experimental efforts focused on revealing the molecular machinery involved in antigen recognition events and led to the formulation of concepts implicating kinetic segregation, kinetic proof-reading, co-receptor involvement, ligation-triggered conformational changes, serial engagement, and the contribution of mechanical forces in early recognition events [reviewed in  are continuously remodeled by cellular enzymes, (iii) are primarily negatively charged, and (iv) play an important role in cellular processes such as signal amplification, adhesion, migration, and cell death  , 34, andin vivo , this limitation dramatically affected T-cell responsiveness, as only treatment with pMHCs but not with antibodies triggered T-cell activation under these conditions, except for very high ligand densities  and 70–350 nm in diameter with a density of 3–4 protrusions per µmg T cell , 70. So g T cell . This cog T cell –73 have Microvilli were described as continuously forming under the leading edge of the lamellipodium of migrating T cells. Upon antigen encounter and synapse formation, membrane protrusions can be preferentially observed forming at the synaptic periphery of the T cell–APC interface . ImportaMonitoring and characterizing of T cell microvilli during antigen scanning remains a big challenge within the otherwise extensively researched field of T-cell activation. Previous investigations are confounded by varying cell types, T-cell receptors, and presented ligands, but ultimately by the applied techniques and stimulation platforms. Studies were especially hindered by the lack of methods to investigate three-dimensional and highly dynamic nanostructures on living cells . A few rAs extensively reviewed by Orbach and Su , microviIn summary, it is evident that microvilli form a restricted reaction volume harboring the necessary molecules for T-cell activation. An exciting observation was recently made by Klotzsch and colleagues, who demonstrated the ability of the T cell to reach into narrowly confined spaces and who showcased the very dynamic nature of their microvillar protrusions and their ability to scan for occluded antigens . Interes+ T cells move with a mean velocity of ~3 µm/min over cell surfaces . Perpenvia microvilli in antigen discrimination. The suggested mechanism is in agreement with the finding that microvilli stabilization is independent of actin . Although the depth of these structures was found to be stimulus-independent, the width showed a significant decrease in the presence of a specific antigen . The autof actin : Within in vivo , Zhu and colleagues found a correlation between the bond lifetime and stimulatory potency , 129. BF+  and CD4+ T cells , 123, an T cells , catch-b T cells . Therefo T cells , 122. Itut cells .Adhesion-related forces of about 1–2 nN have been reported within cell–cell contacts. However, measured values varied significantly depending on the cell line under investigation , 132. MoHusson et al.  adapted Traction force microscopy (TFM) has been applied to assess forces related to T-cell adhesion and activation. This methodology involves tracking the force-induced displacement of fluorescent beads embedded in a matrix of defined stiffness resulting from cells crawling or spreading thereon. From such studies, it was concluded that T cells adapt their signaling response according to substrate stiffness. TFM provided the net amplitude and directionality of averaged cellular mechanical forces. With the use of elastic pillars coated with pMHC or activating antibodies, Bashour et al. visualized and quantified TCR-imposed forces : T cellsAtomic force microscopy (AFM) using pMHC-coated cantilevers confronting the T cell surface showed distinct pushing and pulling phases without further application of external force . The net+ T cells were shown to unzip pMHC-carrying hairpins of 12 pN but not of 19 pN resistance, and CD4+ T cells unzipped hairpins of 4.7 pN resistance (Intra- and extracellular molecular force sensors (MFSs) are recent technological additions to the field of nano-mechanosensors and can either have an analog (for peptide/PEG-based sensors) or digital readout (for DNA-based sensors). By defining the mechanical force necessary to unzip a DNA hairpin spanned between a fluorescent dye and a quenching gold particle, Salaita and colleagues successfully measured the range of forces exerted by a defined number of TCR. CD8sistance , 135. Insistance .The shortcomings of digital MFSs can be mitigated by using peptide-based analog MFSs. These contain a flexible peptide whose extension is a continuous function of the applied force. Like a macroscopic spring, the higher the pulling force is exerted, the larger the end-to-end distance of the peptide becomes, allowing for direct measurement of the force. This approach has been showcased by the quantification of forces exerted by single integrins by Morimatsu et al. in 2013 . ReasonaUsing an analog peptide-based MFS, we recently quantified the mechanical forces exerted by single TCRs and found a striking difference between activating and scanning conditions . We deveThe impact of the orientation of the force vector on mechanotransduction is another hotly debated topic in the immune surveillance and T-cell triggering fields. The aforementioned experiments using optical tweezers to pull a bead tangentially or perpendicularly with respect to the T cell surface  yielded via co-stimulation  Microvilli are important for fast and efficient antigen scanning and sampling.(ii) Microvilli carry all molecules necessary for adhesion and initiating TCR-proximal signaling.(iii) Microvilli form limited reaction volumes, which may sensitize T cells for antigen.(iv) Microvilli may not only be involved in the biochemical probing of the environment but may also be necessary for testing the biomechanical properties of the target cells and tissues.(v) Glycocalyx and cellular stiffness parameters affect TCR triggering.sine qua non for further insights in the field, and based on the aforementioned studies, a number of conclusions can already be drawn:Considering the dynamic cellular processes surrounding mechanosurveillance, a precise temporal control of the triggering event is a (i) Tensile forces do affect early T cell activation.(ii) Force transduction is TCR- and peptide-dependent.(iii) Shear forces or torque affect T cell activation more strongly than normal forces.(iv) Generated tensile forces are invariably tied to physiological T cell recognition and precede T cell activation.(v) Individual molecules at protrusion tips are subject to pulling and pushing forces in the pN range.(vi) Tensile forces are generated internally by rearrangements of the actin cytoskeleton and do not involve the action of acto-myosin motor proteins.(vii) Immune synapse formation generates mechanical forces that activate ligand-bound integrins.(viii) T cell-imposed forces deform target cells and promote their killing.(i) Are there any lateral movements of antigen-bound microvilli tips? What resistance does the glycocalyx pose to the lateral movement of microvilli? Can the microvillar scanning process be imagined more like a “dragging through the waves” or more like a repeated “poking through the barrier”?(ii) There is conflicting evidence for increased microvilli life- or dwell times upon antigen encounter. Does antigen binding lead to an arrest of microvilli dynamics? Are such arrests only happening for triggering interactions or also for probing events?(iii) Is the glycocalyx posing a resistance to microvilli penetration?(iv) At what point is the force vector reversed from pushing to pulling? How are adhesive interactions influencing this process? Is this process used for ligand discrimination by mechanical probing?(v) What is the fate of microvillar protrusions after TCR triggering? Are the protein platforms at the microvillar tips evolving to microclusters? What is the impact of LFA-1 maturation on signaling within microvillar protrusions?(vi) Is the mobility of the pMHCs decisive for the interaction probability and/or rebinding in the context of microvillar scanning? Are target cells mechanically altering their presenting surfaces to allow or inhibit efficient scanning of the surface?(vii) How are microvilli recognition events coupled to the movement of the entire cell body? How are these signals communicated within the T cell?JG conceived and drafted the manuscript. JG and LS conducted literature review and wrote the manuscript. LS designed the figures. JG, LS, GS, and JH revised the manuscript. All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.This work was supported by the Austrian Science Fund (FWF) project P32307-B , and I5056-B (LS and GS), and by the Vienna Science and Technology Fund (WWTF) project LS13-030 .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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.