In January 2017, the Dutch cervical cancer screening programme transitioned from cytomorphological to primary high-risk HPV (hrHPV) DNA screening, including the introduction of self-sampling, for women aged between 30 and 60 years. The Netherlands was the first country to switch to hrHPV screening at the national level. We investigated the health impact of this transition by comparing performance indicators from the new hrHPV-based programme with the previous cytology-based programme.We obtained data from the Dutch nationwide network and registry of histo- and cytopathology (PALGA) for 454,573 women eligible for screening in 2017 who participated in the hrHPV-based programme between 1 January 2017 and 30 June 2018  and for 483,146 women eligible for screening in 2015 who participated in the cytology-based programme between 1 January 2015 and 31 March 2016 (maximum follow-up of 40 months). We compared indicators of participation (participation rate), referral  and detection  detection; number of referrals per detected CIN lesion).Participation in the hrHPV-based programme was significantly lower than that in the cytology-based programme (61% vs 64%). Screen positivity and direct referral rates were significantly higher in the hrHPV-based programme . CIN2+ detection increased from 11 to 14 per 1000 women screened. Overall, approximately 2.2 times more clinical irrelevant findings (i.e. ≤CIN1) were found in the hrHPV-based programme, compared with approximately 1·3 times more clinically relevant findings (i.e. CIN2+); this difference was mostly due to a national policy change recommending colposcopy, rather than observation, of hrHPV-positive, ASC-US/LSIL results in the hrHPV-based programme.This is the first time that comprehensive results of nationwide implementation of hrHPV-based screening have been reported using high-quality data with a long follow-up. We have shown that both benefits and potential harms are higher in one screening round of a well-implemented hrHPV-based screening programme than in an established cytology-based programme. Lower participation in the new hrHPV programme may be due to factors such as invitation policy changes and the phased roll-out of the new programme. Our findings add further to evidence from trials and modelling studies on the effectiveness of hrHPV-based screening. Primary high-risk HPV (hrHPV) DNA screening, evaluated in clinical trials, has been shown to be more effective and cost-effective than cytology screening for the detection of pre-malignant and malignant cervical lesions , 2. FollAs part of the initial feasibility study, modelling analysis was conducted assessing the costs and effects of implementing primary hrHPV-based screening in the Netherlands . Recent The success of a screening programme depends on the implementation of well-defined protocols and guidelines . ScreeniData from the Dutch nationwide network and registry of histo- and cytopathology (PALGA) has enabled regular, high-quality monitoring of organised cervical cancer screening in the Netherlands for many years. This comprehensive dataset has national coverage , enablinUntil the end of 2016, the Dutch cervical cancer screening programme used cytology as the primary screening test. Women were invited to make an appointment for screening with their GP every 5 years from ages 30 to 60. Women could choose to opt out of screening either temporarily  or indefinitely .There were various referral pathways in the cytology-based programme, depending on the result of primary cytology screening Fig. a. DirectPrimary hrHPV screening was implemented in the Netherlands on 1 January 2017  were referred for colposcopy, while hrHPV-positive women with normal cytology were invited for repeat cytology testing after 6 months.In the Netherlands, there are five regional screening organisations responsible for the implementation of the screening programme. With the change from cytology-based to hrHPV-based screening, the policy for inviting women was changed, with the regional screening organisations sending all invitations in a standard manner; women were all invited after their birthday in the year they were eligible for invitation. In the cytology-based programme, invitations were sent by the regional screening organisation, GP practices or using a combined approach. The timing of the invitation also varied depending on which organisation sent the invitation; some invitations were sent at the start of the year that women would become eligible to participate, and some were sent after the women’s birthdate. The number of laboratories responsible for analysing primary screens from the programme was reduced from approximately 40 in the cytology-based programme to five in the hrHPV-based programme (one per region).Clinician-collected samples were collected in 20 ml ThinPrep medium , transported and stored at room temperature until processed in the laboratory. The Evalyn® Brush  was used for self-sampling. The self-collected brushes were sent to the laboratories by regular mail. The brush of the self-sampling device was transferred into 20 ml of ThinPrep medium prior to hrHPV testing. All laboratories used the cobas® 4800 HPV test  to test the clinician-collected and self-samples. The cobas® 4800 HPV test is a CE in vitro diagnostic (IVD) certified kit  for use in combination with the cobas® 4800 system for nucleic acid extraction, PCR setup, real-time PCR amplification and result analysis. As part of the assay procedure, each sample was also tested for the presence of human cells by amplification of the human beta-globin gene. The clinical performance of the cobas® 4800 system has been validated using Dutch samples , and theThis study is a longitudinal, retrospective population-based cohort study. We obtained results of primary screening tests and any associated follow-up from the Dutch nationwide network and registry of histo- and cytopathology (PALGA) for two cohorts. The cytology cohort consisted of women who participated in the cytology-based screening programme between 1 January 2015 and 31 March 2016 (maximum follow-up of 40 months). The hrHPV cohort consisted of women who participated between 1 January 2017 and 30 June 2018 in the hrHPV screening programme . An inclusion period of 18 months was used for the hrHPV cohort to compensate for the phased implementation of the new programme , referral , referral rate from follow-up smear  and total referral rate ) and detection .To compare the performance of the hrHPV-based screening programme with the cytology-based screening programme, we calculated indicators in three categories: The participation rate was defined by the number of primary screening tests divided by the number of women eligible for screening. The number of eligible women was estimated from the number of women in the Dutch population who would reach screening age in 2015 or 2017  on 1 January 2015 for the cytology cohort and on 1 January 2017 for the hrHPV cohort. This data was obtained from Statistics Netherlands  and adjuReferrals were identified based on advice codes recorded in PALGA and could be direct or indirect . The participation rate in the hrHPV-based programme was lower in all age groups. The biggest difference was found in age group 45–49 years . Differences in participation rates were statistically significant for all age groups (p < 0.001).Figure p < 0.001).The percentage of inadequate cytology smears recorded at primary screening as a proportion of all primary screening reduced from 1.6% in 2015 to 0.1% in 2017  . Related to this, we found that the proportion of women referred to the gynaecologist also significantly increased . The increases in positive screen tests and in the referral rate were largest in women aged 30–34 years, where the proportion of positive screen tests increased from 9% in the cytology-based programme to 21% in the hrHPV-based programme (p < 0.001) and the referral rate increased from 3% to 8% (p < 0.001).Figure p < 0.001). In addition, amongst hrHPV-positive women, more women had a cytological abnormality after self-sampling than clinician-collected sampling  . Overall, the referral rate doubled and the CIN2+ detection rate increased by 34% (p < 0.001). For the youngest age group, the referral rate increased by 92% (p < 0.001) and the CIN2+ detection rate by 30% (p < 0.001).Figure p = 0.974), cytology or histology was performed. The CIN2+ detection rate after cytology or histology varied across the four different groups in the hrHPV-based programme: from 35.7% in indirect referred women after a clinician collected sample to 57.1% in direct referred women after self-sampling . Screen positivity was higher in the hrHPV-based programme. The cytology programme recommended observation of ASC-US/LSIL results, while the hrHPV-based programme recommended colposcopic referral for hrHPV-positive, ASC-US/LSIL results. As expected, this increased both the number of colposcopic referrals and CIN2+ lesions detected.The introduction of self-sampling had been expected to increase participation, as a previous Dutch study (PROHTECT) found that screening non-attenders who were offered self-sampling were more likely to be screened than non-attenders . While 8Organisational factors, such as the phased roll-out of the new programme and changes in the invitation process, may also have resulted in lower participation. Due to the phased roll-out of the new programme over the first quarter of 2017, women had less time to take up their screening invitations compared with the cytology-based programme, although we still observed a lower participation rate when calculating it based on 18 months of data. If the phased implementation is the cause of lower participation, we would expect participation to increase in the coming months. In the cytology-based programme, GP practices could invite patients for screening, rather than women receiving an invitation from the regional screening organisation. Women who received invitations sent from GP practices were more likely to participate in the cytology-based programme than women who received invitations from screening organisations . DiscontThe hrHPV positivity rate was higher than anticipated at 9.1%, as a previous population-based Dutch study (DuSC) found a hrHPV positivity rate of 8% amongst women of screening age . This diOne surprising finding was that hrHPV positivity was lower in self-samples than in the clinician-collected samples, contrary to previous Dutch studies. One population-based study found higher hrHPV positivity in self-samples than in clinician-collected samples , and oneA higher CIN2+ detection rate was found in the hrHPV programme than in the cytology-based programme. This was expected based on the results of four large randomised trials of HPV screening . HoweverIn several countries, hrHPV-based screening has been implemented, but published results are only available from Italy and Turkey. In Italy, HPV-based screening was implemented in 2012 in 19 screening programmes across ten regions. The direct referral rate from the Italian programme was comparable with the Dutch programme at 2.9% . In 2014Given the increased sensitivity of hrHPV testing for CIN2+ lesions, detection rates are expected to be higher in the first round, as both prevalent and incident lesions are detected. As the programme reaches a steady state, and fewer prevalent lesions are detected, we expect that detection of CIN3+ lesions will decrease, as seen in the POBASCAM trial . TherefoThis is the first study to report the results of the nationwide implementation of a hrHPV-based screening using prospectively collected cyto- and histopathological data. We have been able to compare this reliably with the previous cytology-based programme due to the nationwide coverage of PALGA. The large number of screens included in our study has allowed us to make statistically robust comparisons between indicators of the two programmes. Our study has some limitations. The follow-up time included in our study was shorter for the hrHPV-based programme than for the cytology-based programme, as the hrHPV-based programme was implemented more recently. We are unable to analyse characteristics of non-attenders to the programme, as characteristics of these women are not captured by PALGA. We are also unable to differentiate loss to follow-up after referral for colposcopy from cases where women attended colposcopy, but no cytology or histological diagnostic test was performed. This information is unavailable for both the hrHPV-based programme and the cytology-based programme. As such, we cannot investigate whether adherence to referral advice has changed over time. Furthermore, compliance to referral, used to differentiate cytology only and no follow-up with cytology or histology in Table This is the first time that results of nationwide implementation of hrHPV-based screening have been reported using high-quality data with extended follow-up. Our results show implementation of the hrHPV-based programme has been successful. However, the lower participation rate in the hrHPV-based programme needs to be investigated further to ensure that the screening programme remains effective and efficient. Detection of CIN2+ lesions was higher in the hrHPV-based programme at the cost of more unnecessary referrals. Careful consideration needs to be given to potentially changing triage of hrHPV-positive screens to reduce unnecessary referrals. Ongoing monitoring of the hrHPV-based programme is essential to ensure that a reasonable balance of benefits and harms continues to be achieved.Additional file 1. Detailed description of methods for calculating results.Table A1. Age groupings used in analysis by programme type. Table A2. Calculation of the indicators shown in Figure Table A3. Calculation of the indicators for participation, referral and detection in the old cytology-based screening programme, cohort 2015, and within the new hrHPV-based screening programme, 2017 cohort.


Seed-based whole-brain FC analysis revealed that brain regions with decreased FC with the contralateral S1M1 extended beyond the sensorimotor network to the prefrontal and visual cortices . Moreover, correlation analysis showed that decreased FC between the subcortical and the cortical regions in the sensorimotor network progressively increased in relation to the time since amputation. These findings indicated a cascade of cortical reorganization at a more extensive network level following lower-limb amputation, and also showed promise for the development of a possible neurobiological marker of changes in FC related to motor function recovery in LLAs.Cerebral neuroplasticity after amputation has been elucidated by functional neuroimaging. However, little is known concerning how brain network-level functional reorganization of the sensorimotor system evolves following lower-limb amputation. We studied 32 unilateral lower-limb amputees (LLAs) and 32 matched healthy controls (HCs) using resting-state functional magnetic resonance imaging (rs-fMRI). A regions of interest (ROI)-wise connectivity analysis was performed with ROIs in eight brain regions in the sensorimotor network to investigate intra-network changes, and seed-based whole-brain functional connectivity (FC) with a seed in the contralateral primary sensorimotor cortex (S1M1) was used to study the FC reorganization between the sensorimotor region (S1M1) and other parts of the brain in the LLAs. The ROI-wise connectivity analysis showed that the LLAs had decreased FC, mainly between the subcortical nuclei and the contralateral S1M1 ( With increases in traffic accidents, malignant diseases and natural disasters, the number of lower-limb amputees (LLAs) is increasing . LLAs haOne of the most prominent consequences of limb amputation concerns the functional reorganization in the primary somatosensory and motor cortices. Several studies in non-human primates have demonstrated that the loss of an upper limb results in functional reorganization of the primary sensorimotor cortex (S1M1) . In humaAlthough emphasis has been given to the sensorimotor cortex in the functional reorganization following limb amputation, the contribution of subcortical structures cannot be overlooked . The cerIn recent years, functional connectivity (FC) analyses have provided invaluable approaches for studying the human brain in healthy  and diseBesides the regions within the sensorimotor network, abnormal brain reorganization has been found in other regions . SpecifiAccording to the research mentioned above, amputation may result in extensive structural and functional reorganization in the brain. However, very little is known about large-scale network-level reorganization of FC following lower-limb amputation, which requires further investigation. Here, we take these ideas further by characterizing intra-network (within the sensorimotor network) and inter-network (between S1M1 and other parts of the brain) changes following lower-limb amputation using resting-state FC analysis. We hypothesized that the LLAs would show abnormal FC within and beyond the sensorimotor network compared with the healthy controls (HCs); meanwhile, when considering the long duration of recovery for some amputees, we also hypothesized that FC in the LLAs may present progressive changes in relation to the time since amputation. To test these hypotheses, we compared the regions of interest (ROI)-wise connectivity within the sensorimotor network and the seed-based whole-brain FC of the sensorimotor network between the LLAs and HCs. Further, we investigated the relationship between FC and time since amputation in LLAs.Table 1) were recruited through the Prosthetic and Orthotic Clinics at the Department of Rehabilitation, Southwestern Hospital  between March 2015 and April 2016. Nineteen amputations occurred at the transfemoral level and 13 at the transtibial level. All the patients had been fitted with prostheses. Twenty-five patients underwent amputation following a traumatic injury, and the other amputations were due to tumors . Phantom limb pain (PLP) was assessed using a 10 cm Visual Analog Scale (VAS)  with acquired unilateral lower-limb amputation  was used to acquire the functional and structural data. Rs-fMRI data were collected via a multi-slice, gradient-echo, echo-planar sequence for a total of 240 volumes (480 s) using the following parameters: repetition time (TR) = 2000 ms; echo time (TE) = 30 ms; flip angle = 90°; matrix = 64 × 64; field of view = 200 mm; slice thickness = 3 mm, 1-mm gap, 36 slices with a voxel size = 3 mm × 3 mm × 3 mm. During the resting-state scan, subjects were asked to relax with their eyes closed and not to think of anything in particular. High-resolution three-dimensional T1-weighted structural images were also collected in the sagittal orientation via a magnetization-prepared rapid gradient-echo sequence (MP-RAGE) using the following parameters: TR = 1,900 ms, TE = 2.52 ms, flip angle = 9°, matrix = 256 × 256, thickness = 1.0 mm, 176 slices with a voxel size = 1 mm × 1 mm × 1 mm.Before data preprocessing, in order to obtain a ‘homogeneous’ sample of patients with a left side amputation, we flipped the imaging data from right to left along the midsagittal line for the 13 patients with an amputation on the right side. For all patients, the left side corresponded to the cerebral hemisphere ipsilateral to the side of the amputation and the right side corresponded to the cerebral hemisphere contralateral to the side of the amputation.1. The first 10 images of each subject were discarded to allow the signal to reach equilibrium and the participants to adapt to the scanning noise. Then, the remaining 230 images were corrected for temporal delay in slice acquisition and co-registered to the first image for correction of rigid- body head motion. For all subjects, the translation or rotation parameters did not exceed ± 2.5 mm or ± 2.5°, respectively. Since rs-fMRI data have been demonstrated to be sensitive to head motion (Preprocessing was performed using the Data Processing Assistant for Resting-State fMRI (DPARSF) toolboxd motion , the head motion . To furtd motion . The res2 . For simplicity, in the following sections, the brain region contralateral to the side of the amputation was called the contralateral region, and the brain region ipsilateral to the side of the amputation was called the ipsilateral region in LLAs. For example, the S1M1 contralateral to the side of the amputation was called the contralateral S1M1. ROI-wise connectivity was measured by extracting the residual time course data from each ROI listed above and calculating the Pearson’s correlation coefficients between each ROI pair. The resulting correlation coefficients were then Fisher-transformed into ’Z’ scores to increase normality.For ROI-wise connectivity, we selected eight subcortical and cortical sensory- and motor-related brain regions based on a freely available atlas of regions defined by correlated activation patterns2 , includi2  has demo ganglia . All thenglia  were performed to compare the demographic and clinical data from the two groups . The significance level was set at p < 0.05.Two-tailed two sample 5. Two-tailed two-sample t-tests were conducted to identify the specific group differences . Age, sex, and years of education were controlled as covariates to ensure that our results only revealed FC differences uniquely associated with amputation.Analyses of the ROI-wise connectivity were performed using the GRETNA toolboxt-tests were performed to examine group-related differences between the LLAs and the HCs. Significance thresholds for t-test were set at p < 0.05 (FDR corrected). The covariates age, sex, and educational level were controlled. These statistical analyses were carried out with the SPM toolbox (spm86).For the FC maps (seed-based whole-brain voxel-wise connectivity), two-tailed two-sample p < 0.05).We correlated clinical characteristics with brain FC, corresponding to the significant group-related difference between the LLAs and the HCs. A two-tailed partial correlation analysis was used after controlling for age, sex, educational level as confounding variables . PLP was present in 12 LLAs [ranging in pain intensity from 1.1 to 6.8 (3.46 ± 2.19)].p < 0.05, FDR corrected) .For ROI-wise connectivity, compared with the HCs, the LLAs exhibited significantly decreased FC between the contralateral S1M1 and the bilateral basal ganglia, bilateral thalamus, and cerebellum, between the cerebellum and the bilateral basal ganglia and bilateral thalamus, and between the SMA and the ipsilateral thalamus and contralateral basal ganglia  and the ipsilateral occipital lobe in LLAs  .The seed-based analysis provides an opportunity to validate and extend the main results obtained in the ROI-wise connectivity analysis, as it allows us to identify which specific loci might underlie the changes in FC between the contralateral S1M1 and the brain regions within and beyond the sensorimotor network in LLAs. When comparing connectivity maps between amputees and the HCs, significantly decreased FC was found between the contralateral S1M1 seed and the ipsilateral cerebellum and the ipsilateral thalamus in the LLAs, which is consistent with the results of the ROI-wise connectivity analysis described above. In addition, the seed-based map in To test for possible hemisphere-specific effects, all the calculations were performed on the patients with only right- or left-sided amputations separately. The results did not reveal hemisphere specific differences and therefore all the patients were included in one calculation with a virtual left side amputation in this study. Furthermore, to detect the possible effects of the PLP, we divided all the patients into two groups (LLA with PLP and LLA without PLP). We compared the ROI-wise and the seed-based FC between two groups and failed to find any significant differences between the two groups.Figure 4, partial correlation analysis revealed that the FC between the SMA and the contralateral basal ganglia and ipsilateral thalamus and between the contralateral S1M1 and the contralateral basal ganglia and ipsilateral thalamus was positively correlated with the time since amputation in LLAs . Only the correlations between the time since amputation and FC between the SMA and the contralateral basal ganglia and ipsilateral thalamus survived after FDR correction for multiple comparisons . Meanwhile, partial correlation analysis showed that the extracted FC of the cluster  identified in the group comparison of seed-based FC was significantly correlated with the time since amputation in LLAs .For ROI-wise connectivity, as shown in Accumulating evidence has indicated that amputation induces functional reorganization of the sensorimotor areas in the brain, including the S1M1, cerebellum, thalamus, and basal ganglia, which could be related to abnormal functioning of the subcortical-cortical sensorimotor system resulting from the reduction or absence of afferent information following amputation . In addiIn the current study, we investigated changes in the organization of the intra- and inter-FC of the sensorimotor network in unilateral LLAs, and the relationship between FC and the time since amputation. We identified abnormal FC within and beyond the sensorimotor network. In addition, we found positive correlations between FC among subcortical and cortical sensorimotor regions and the time since amputation.As a drastic limb injury, amputation in humans induces changes in the S1M1 of the deafferent hemisphere . In thisIn addition to cortical areas, the sensorimotor network consists of subcortical areas such as the cerebellum, basal ganglia and thalamus . A previThe seed-based analysis also revealed aberrant connectivity within the sensorimotor network in LLAs. We found that the FC between the contralateral S1M1 and the ipsilateral cerebellum and the ipsilateral thalamus was reduced. These results confirmed the functional reorganization of the sensorimotor network in the patients with lower-limb amputation. In addition, we found reduced FC between the contralateral S1M1 and the ipsilateral superior frontal gyrus  in the LLAs. The superior frontal gyrus  is a part of the DMN , which iOur correlation analyses revealed statistically significant positive associations between the time since amputation and the FC in the contralateral basal ganglia, ipsilateral thalamus and SMA, and the contralateral basal ganglia, ipsilateral thalamus and contralateral S1M1 in the LLAs, which suggested that there were progressive subcortical-cortical FC changes in LLAs. Recent MRI studies have demonstrated that the brain changes over time following amputation . HoweverDespite of the important results showing brain functional network reorganization in the LLAs mentioned above, several limitations in this study should be further considered. First, in the current study, we mainly choose the sensorimotor network to study the functional reorganization process over time since amputation, which limits our ability to speculate on global brain organization patterns after amputation. Second, the sample size of the LLAs is relatively small. Studies with larger sample sizes are needed to clarify the specific functional reorganization mechanisms in patients with different types of amputations and to disentangle the influences of the amputation site, the amputation side and the use of prostheses on cortical reorganization in amputees. Third, previous studies found that PLP may affect brain functional reorganization  and coulOur results revealed that lower-limb amputation leads to subcortical and cortical reorganization in the sensorimotor network. Moreover, between-group differences in the seed-based whole-brain FC analysis showed that brain regions with decreased FC with the contralateral S1M1 extended to the prefrontal [superior frontal gyrus ] and occipital cortices. These findings suggested that functional reorganization after lower-limb amputation occurred at multiple scales, within and beyond the scope of the sensorimotor network. Finally, correlation analysis results showed that decreased FC between the subcortical and the cortical regions  progressively increased over the time since amputation, which reflects the process of brain functional network recovery, probably related to motion exercises or prosthesis use after amputation. All of these results hold promise for developing reliable biomarkers of FC changes related to motor function recovery in LLAs.We declare that all human studies have been approved by the Army Medical University Ethics Committee and have therefore been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. We declare that all patients gave informed consent prior to inclusion in this study.JZ designed and performed the experiments, analyzed the data, and wrote the manuscript. LW and LS assisted in fMRI data analysis. YZ assisted in writing and editing the manuscript. LL and PL assisted in sample collection and clinical data analysis. XY and MQ provided overall project supervision, helped to analyze and interpret the data, and assisted in writing the manuscript. All authors have read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer MS and handling Editor declared their shared affiliation.


Active meiotic chromosome movements are a universally conserved feature. They occur at the early stages of prophase of the first meiotic division and support the chromosome pairing process by (1) efficiently installing the synaptonemal complex between homologous chromosomes, (2) discouraging inadvertent chromosome interactions and (3) bringing homologous chromosomes into proximity. Chromosome movements are driven by forces in the cytoplasm, which are passed on to chromosome ends attached to the nuclear periphery by nuclear-membrane-spanning protein modules. In this extra view, we highlight our recent studies into the role of the nuclear lamina during this process to emphasize that it is a highly conserved structure in metazoans. The nuclear lamina forms a rigid proteinaceous network that underlies the inner nuclear membrane to provide stability to the nucleus. Misdemeanors of the nuclear lamina during meiosis has deleterious consequences for the viability and health of the offspring, highlighting the importance of a functional nuclear lamina during this cell cycle stage.Abbreviations: DSB: DNA double strand break; LEM: LAP2, Emerin, MAN1; LINC: LInker of the Nucleoskeleton and Cytoskeleton; RPM: rapid prophase movement; SUN/KASH: Sad1p, UNC-84/Klarsicht, ANC-1, Syne Homology Haploid gametes are produced as a consequence of meiotic cell divisions, and genetic diversity is generated through the assortment and exchange of parental chromosomes via the programmed induction of DNA double strand breaks (DSBs). DSB repair through homologous recombination creates a physical connection between the parental homologous chromosomes, which promotes their proper segregation. Chromosome segregation requires their side-by-side alignment, which is ultimately stabilized by establishment of the synaptonemal complex . In all Caenorhabditis elegans, the LINC complex consists of SUN-1 and ZYG-12, located in the inner and outer nuclear membranes, respectively. The N-terminus of ZYG-12 extends into the cytoplasm and interacts with dynein, thereby connecting to the microtubule cytoskeleton, whereas the N-terminus of SUN-1 resides in the nucleus and cooperates with chromosome domains called pairing centers, which couple the nuclear envelope-associated chromosome end to the movement apparatus, for review see ), in which all the identified phosphorylated residues flanking the rod domain were substituted by non-phosphorylatable alanines, was more resistant to detergent than the wild type. In these mutants the lamina might become a mesh, that behaves very rigid. With this mutant, we also showed that a more rigid lamina network delays meiotic entry, decelerates chromosome movements, and delays chromosome pairing and DSB repair. In agreement with the hypothesis that a lack of phospho-modification would prevent de-crosslinking of the lamina, we found that nuclear lamina disassembly is delayed during the first embryonic division in the [8Agfp::lmn-1] mutant, We showed that upon meiotic entry the amount of peripheral lamin is reduced and it becomes more sensitive to detergent, thus rendering it more ‘soluble.’ The increased solubility depends primarily on the meiotic kinases CHK-2 and (to a lesser extent) PLK-2, since both 8A]; ced-3(n717)gfp::lmn-1 mutant, there was a significant increase in embryonic lethality (roughly a quarter of the embryos die) and diakinesis oocytes with abnormal chromosome structures can be seen. Thus, we concluded that a more rigid lamina network causes stochastic chromosome damage in the prophase of meiosis I, and that the resultant compromised oocytes are efficiently culled by the apoptotic machinery.Surprisingly, these phenotypes do not have a strong impact on the viability of the mutant or on the timing of nuclear envelope breakdown in mitosis under laboratory conditions. However, we noticed increased germline apoptosis in those mutants with a more rigid lamina network. In fact, when the apoptotic machinery was deactivated in the [8A];[sun-1::gfp10Agfp::lmn-1] double mutant. Strikingly, we observed abnormal chromosome structures such as entanglements and chromosomal interlocks in pachytene, a phenotype that is never observed in the wild type or any of the single mutants. These structures persisted until diakinesis and were associated with a significant increase in offspring lethality, for a summary see In a previous study, we identified a meiotic surveillance system mediated by SUN-1 phosphorylation. If meiosis is dysfunctional, then SUN-1 phosphorylation persists to extend chromosome movement and delay meiotic progression, thus providing more time for error correction . By reasLink et al. 2018) highlight several important aspects of lamina network function in meiosis 8 highlig lamina-mC. elegans germline. Phosphorylation and de-crosslinking of the lamina network on the nuclear rim coincides with the appearance of RPMs. When the lamina crosslinking (and thus its rigidity) is artificially maintained, as in the [8Agfp::lmn-1] mutant, chromosome movements are slower. By measuring the ability of chromosomes to diffuse along the nuclear envelope (by preventing force transmission through the cytoplasm), we confirmed that the more rigid lamina in [8Agfp::lmn-1] restricts chromosome end diffusion [8Agfp::lmn-1] mutant could be explained by the cytoskeletal forces being strong enough to overcome hindrance by the more rigid lamina. In addition, the more rigid lamina did not impair the interaction of chromosome ends with the nucleoplasmic component of the SUN/KASH bridge. Overall, the lamina is not eliminated, when RPMs take place, which supports the notion that the lamina itself does not impede chromosome end attachments. Lamin filaments are thinner than regular intermediate filaments [8Agfp::lmn-1] mutant is a hypomorph, thereby having a less severe phenotype. However, we suspect that lamina rigidity is also mediated by many other lamina- and nuclear envelope-associated proteins, and therefore that lamina remodeling might be a multifactorial process. Although the lamina clearly persists for longer in mitotic cell divisions in the mutant  mutant. In this study, however, we identified two mechanisms for preventing the segregation of fused/entangled chromosomes into oocytes: apoptosis and the prolongation of the movement process. In the : they might be caused by either delayed chromatin organization or less-efficient chromosome movement in the [ild type . Howeverngements . These eC. elegans, led to the identification of the homology recognition/pairing center regions, for review see [Regarding the third point of the lamina-modifying kinases concentrated at the subtelomeric chromosome regions; experiments using the tractable genetic model, view see . These aview see ,36. Pairview see . They acview see –40. Pairview see . InteresAt first sight, the pairing centers seem to be an exotic worm-specific feature; however, they cooperate with highly conserved signaling kinases, such as CHK-2 and polo kinases. A role for those in other metazoans during chromosome movements has not been demonstrated to date. The discovery that these specific chromosome domains are involved in nuclear lamina modifications highlights the importance of studying such highly complex processes in relatively simple model organisms. It will therefore be interesting to investigate whether similar modules regulating a crosstalk between chromosome events and the nuclear lamina are also at work in higher eukaryotes.


Pseudoexfoliation (PEX) syndrome is an age-related systemic disease with ocular manifestations. The development of animal models is critical in order to elucidate the cause of the disease and to test potential treatment regimens. The purpose of this study is to report phenotypes found in mouse eyes injected with Adenovirus coding Wnt5a. Some of the phenotypes resemble those found in PEX patients while others are different.Recombinant Adenovirus coding Wnt5a or green fluorescent protein (GFP) were injected into mouse eyes. Two months after the injection, eyes were examined for PEX phenotypes using slit lamp, fluorescence stereomicroscope, histological staining, immunostaining and transmission electron microscope.Certain ocular features of PEX syndrome were found in mouse eyes injected with recombinant Adenovirus coding Wnt5a. These features include accumulation of exfoliation-like extracellular material on surfaces of anterior segment structures and its dispersion in the anterior chamber, saw-tooth appearance and disrupted basement membrane of the posterior iris pigment epithelium, iris stromal atrophy and disorganized ciliary zonules. Ultrastructure analysis of the exfoliation material revealed that the microfibril structure found in this model was different from those of PEX patients.These features, resembling signs of ocular PEX syndrome in patients, suggest that new information obtained from this study will be helpful for developing better mouse models for PEX syndrome. Pseudoexfoliation (PEX) syndrome is an age-related systemic disease characterized by the accumulation of an extracellular fibrillar material in the eyes, skin, lungs, heart, kidneys and other organs ,2. PEX sOver the past 20 years, several theories have emerged to explain the etiology of the disease. First, PEX syndrome has been described as a fibrillinopathy since PELOXL1) were found in 99% of affected patients. This was the first gene found to be associated with PEX syndrome [LOXL1 with PEX syndrome in multiple populations have also observed a high frequency of risk alleles in the general population[LOXL1 is necessary, but not sufficient to cause PEX syndrome. These observations suggest that transgenic mice harboring human risk alleles of LOXL1 alone may not lead to disease manifestation and that screening for other genetic and environmental modifiers is required.Genetic analysis in the Icelandic population revealed that two single nucleotide polymorphisms of the lysyl oxidase-like 1 gene  gene [Currently, only one mouse line exhibits certain features of human PEX syndrome. The mice harbor a spontaneous mutation in the lysosomal trafficking regulator (ST) gene . LYST miin vivo [in vitro [We have previously reported that the activation of noncanonical Wnt signaling is associated with a glaucomatous phenotype in vivo  and in vin vitro . There ain vitro . On the in vitro . This trin vitro . Subsequin vitro . Repressin vitro , but it in vitro . When wein vitro . This unUsing light and electron microscopy as well as immunohistochemistry, we observed several hallmarks of PEX syndrome in the eyes of these mice, including accumulation of exfoliation-like extracellular material on surfaces of anterior segment structures and its dispersion in the anterior chamber, saw-tooth appearance and disrupted basement membrane of the posterior iris pigment epithelium, iris stromal atrophy and disorganized ciliary zonules. Ultrastructure analysis of the exfoliation material revealed that the microfibril structure found in this model was different from those of PEX patients. These findings will be helpful for developing better mouse models for PEX syndrome.9pfu, plaque-forming units) of purified adenovirus was slowly injected into the anterior chamber using a Hamilton syringe. Three days after injection, eyes were evaluated with a fluorescent stereoscope and Optical Coherence Tomographic Imaging . Eyes with apparent corneal opacity, blood in the anterior chamber, closed iridocorneal angle or GFP negativity were excluded from further analysis. Most eyes were collected two months (n = 6 each for AdGFP and AdWnt5a group) after injection for histopathologic and immunohistochemical analyses.Full-length mouse Wnt5a cDNA (pLNC Wnt-5aHA) was a gift from Jan Kitajewski (Addgene plasmid # 18032) . This cDAnimal care and use conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. All animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Cincinnati. The mice used in this project were housed in AAALAC approved animal facilities within the University of Cincinnati/College of Medicine. Programs of animal husbandry, preventive medicine, and pre- and post-surgical care have been developed to assure that adequate veterinary care is provided at all times. Complete veterinary, diagnostic, and clinical support services were available.Slit lamp examinations were performed on a modified Topcon slit lamp. A beam splitter (BS7030-TOPCON) was installed in front of the eyepieces. An AccuBeam Video Adapter was attached to the beam splitter. A high definition digital camera was mounted on the Video Adapter. Animals were anesthetized and pupils were dilated, a vertical broad slit-lamp beam was placed on the pupillary margin. Still images were taken under the same beam intensity and exposure time.Fluorescein Ak-Fluor , diluted with sterile 1 × DPBS ; was administered by bolus injection (50 μL) into the peritoneum of anesthetized mice. For iris angiography, the non-dilated eyes were observed under Axio Zoom.V16 stereo fluorescence microscope from Zeiss .Enucleated eyes were fixed in Davidson`s fixative overnight and dehydrated through graded ethanol steps, and embedded in paraffin according to standard protocols. Five micrometer thick sections were deparaffinized, rehydrated to PBS, and stained with Hematoxylin and Eosin (H&E) or Periodic acid–Schiff (PAS) reagents. To visualize the tissue structure of the iris, a de-pigmentation step was included, prior to staining, by treating the rehydrated slides with 10% hydrogen peroxide in PBS overnight. For immunofluorescence staining, an antigen retrieval step was performed by boiling the slides in citrate buffer for 10 minutes. The following primary antibodies were used: rabbit anti-collagen IV , rabbit anti-LOXL1 , mouse anti-fibrillin . Antibody detection was performed with Cy5 labeled goat anti-rabbit IgG  and goat anti-mouse IgG . Fluorescent images were taken using a Leica confocal microscope under 63x oil objective lens.Electron microscopic examination was performed according to previous publications . BrieflyTo express Wnt5a in the anterior segment, Adenovirus-Wnt5a (Adwnt5a) was injected into the anterior chamber of adult mice. The AdWnt5a also co-expressed the GFP. Adenovirus-mediated gene expression was monitored by fluorescence stereomicroscope in live animals. As shown in Under the slit lamp, the anterior lens capsule of AdWnt5a-injected eyes had more topographic surface irregularities than those of AdGPF-injected eyes compare , such asNext, histopathological analyses were conducted to reveal pathological features of AdWnt5a-injected eyes on the light and electron microscopic level. The anterior chamber of control AdGFP-injected eyes was free of extracellular aggregates by H & E staining , while AThe structure of ciliary zonules can be best revealed by overexposing the H & E images. In the control eyes, the main zonular bundles connected the ciliary processes to the anterior lens surface and fan shaped fine bundles attached to the equatorial lens capsule . In AdWnTo reveal the tissue structure of the heavily pigmented iris, a de-pigmentation step was used prior to H & E staining. In AdGFP-injected eyes, the iris was separated by the strong Eosin-positive dilator muscle. Anterior to the dilator muscle was iris stroma and posterior to the muscle was a double layer of pigmented epithelial cells. In control eyes, the anterior and posterior surface of the iris were rather smooth and well-defined . In AdWnPeriodic acid-Schiff's reaction (PAS) staining highlighting structures with a high proportion of carbohydrates, showed positivity of extracellular material aggregates in the anterior chamber of AdWnt5a-injected eyes in contrast to control eyes . ZonulesTransmission electron microscopy of uninjected eye showed that a region of posterior chamber adjacent to the iris pigmented epithelium was free of microfibrils . LateralNext, immunostaining was used to obtain the expression profile of known components of exfoliation material, such as fibrillin and LOXL1. In the control eyes, positive staining for fibrillin was observed in Descemet’s membrane only, whereas corneal endothelium, corneal stroma and lens capsule were largely negative . In contIn control eyes, weak immunopositivity for LOXL1 could be observed in the non-pigmented ciliary epithelium and zonules , and in Collagen type IV is the major component of basement membrane. In control eyes, collagen type IV could be immunolocalized to the anterior and posterior basement membranes of the iris pigment epithelium and blood vessels in the iris stroma . The posMouse models for PEX syndrome are valuable tools for understanding the molecular pathogenesis of the disease. Currently, the only mouse line which has certain ocular features of PEX syndrome is the LYST mouse, which harbors a spontaneous mutation in the Lysosomal-trafficking regulator (LYST) gene . LYST miFor now, it is unclear how Wnt5a may cause the pseudoexfoliation-like phenotype in our mouse model. From the information about the role of Wnt5a in other diseases, we speculate that the pro-fibrotic effect of Wnt5a may be the culprit. Initially, we found an up-regulation of Wnt5a in the corneal stroma following overexpression of TGF-alpha in the corneal epithelium, which led to secondary angle-closure glaucoma . The samLOXL1 is the most important gene in PEX syndrome pathogenesis, though the precise molecular mechanism of how carriers of high-risk LOXL1 alleles are susceptible to the disease is still unclear. We do not know if LOXL1 plays a role in our mouse model but several interesting observations may be worth noting. In normal mouse eyes, low levels of LOXL1 can be detected in the corneal epithelial cells. In AdWnt5a-injected eyes, LOXL1 protein can be found clustering around the posterior pole of the nucleus of basal corneal epithelial cells. It is important to note that the antibody we used, as is the case with most commercial anti-LOXL1 antibodies, is directed against the propeptide region of the protein because the sequence of the catalytic domain is conserved among all lysyl oxidase isoforms. It is unlikely that the lysyl oxidase activity of LOXL1 towards extracellular matrix proteins plays an important role in PEX syndrome pathogenesis [ogenesis  because ogenesis . The rolAlthough our model recapitulated certain ocular features of human PEX syndrome, several characteristics of PEX syndrome are not found in this model. First, there is no iris transillumination defect in this model, although liberation of pigment granules from the iris pigment epithelium could be seen on electron microscopy. The presence of collagen IV in the basolateral surface of iris pigment epithelial cells suggests the loss of cell junctions. However, the beginning saw-tooth configuration of the iris pigment epithelium may be the preceding event to further loss of pigment and epithelial cells leading to macroscopically evident transillumination defects. Another difference between this model and human PEX is the lack of a typical pattern of exfoliation deposit on the anterior lens capsule, i.e., a translucent central zone of the size of the undilated pupil, a clear intermediate zone and a granular peripheral zone . In our In conclusion, we observed certain ocular features of PEX syndrome in mice with transient Wnt5a expression. There are some common characteristics among human PEX syndrome, LYST mice, and AdWnt5a-injected mice. They are all associated with a pronounced pathology of the iris and production/accumulation of fibrillar extracellular matrix. More studies are needed to examine the potential relationship among lysosomal dysfunction, LOXL1 maturation and Wnt5a activity.S1 FigAdenovirus (GFP/Wnt5a co-expression) was injected into the anterior chamber. The same eye was imaged by the fluorescence stereomicroscope at different time point. Three days after the injection, GFP-positive started showing up mainly in the trabecular meshwork region, forming a “green ring” around the cornea. The injection site was still visible at 4 o`clock region (A). Six days after the injection, more GFP-positive cells were found in the cornea, and later confirmed to be corneal endothelial cells. The injection site was in the process of healing (B). Fourteen days after the injection, GFP signal disappeared from the trabecular meshwork (C) and the injection site was completely healed. Twenty three days after injection, almost all the GFP signal disappeared.(TIF)Click here for additional data file.S2 FigSix days after the Adenovirus (GFP/Wnt5a co-expression) injection, live image was taken by fluorescent stereomicroscope using Z-stack scanning. GFP-positive cells can be found all over the cornea. Cornea in the pupil region should also be positive but was overwhelmed by the reflect green light (A). High magnification revealed the morphology of the GFP-positive cells. Hexagon/star-shaped cells found in the cornea were endothelial cells. Mesenchymal cells found in the TM region were trabecular meshwork cells (B).(TIF)Click here for additional data file.S3 FigAdGFP and AdWnt5a virus were injected into the anterior chamber of mouse eye. Two weeks and two months after injection, the eyes were collected and subjected to anti-Wnt5a immunostaining. Two weeks after injection, no positive signal can be found in AdGFP-injected eye (A). Strong positive signal can be found in the trabecular meshwork and corneal endothelial cells of AdWnt5a-injected eye (B). Two months after injection, AdGFP-injected eye was negative for Wnt5a (C). In AdWnt5a-injected eye, strong positive signal in the endothelial cells disappeared. The overall signal was stronger than that of the control cornea but need to be verified by a secondary detection method such as in situ hybridization.(TIF)Click here for additional data file.


The limited information available on the structure of complexes involving transcription factors and cognate DNA response elements represents a major obstacle in the quest to understand their mechanism of action at the molecular level. We implemented a concerted structural proteomics approach, which combined hydrogen-deuterium exchange (HDX), quantitative protein-protein and protein-nucleic acid cross-linking (XL), and homology analysis, to model the structure of the complex between the full-length DNA binding domain (DBD) of Forkhead box protein O4 (FOXO4) and its DNA binding element (DBE). The results confirmed that FOXO4-DBD assumes the characteristic forkhead topology shared by these types of transcription factors, but its binding mode differs significantly from those of other members of the family. The results showed that the binding interaction stabilized regions that were rather flexible and disordered in the unbound form. Surprisingly, the conformational effects were not limited only to the interface between bound components, but extended also to distal regions that may be essential to recruiting additional factors to the transcription machinery. In addition to providing valuable new insights into the binding mechanism, this project provided an excellent evaluation of the merits of structural proteomics approaches in the investigation of systems that are not directly amenable to traditional high-resolution techniques. A key role in the regulatory of expression machinery is covered by transcription factors (TFs), proteins that recognize target DNA sequences called response elements and establish specific interactions with additional factors to activate or inhibit the transcription process ,2,3,4. TPowered by the development of new experimental strategies and mass spectrometric (MS) instrumentation, structural proteomics has rapidly become an essential approach for gathering valuable structural information for species that are not directly amenable to conventional high-resolution techniques ,12,13,14In this study, we evaluated the concerted application of complementary structural proteomics techniques to overcome the challenges posed by full-fledged complexes between TFs and respective DNA response elements. We selected a model system consisting of the human transcription factor FOXO4 and its cognate Daf-16 family member-binding element (DBE) . FOXO4 iThe experimental strategies were selected for their ability to provide specific information on a typical protein-DNA complex. For instance, HDX was applied to recognize the regions of the protein affected by DNA binding, either through direct protection of the contact interface, or through allosteric conformational changes involving distal regions of the protein. Quantitative XL was used instead to identify possible variations between free and DNA-bound DBD structures, which would help elucidate the effects of the interaction on overall structure topology. In the case of the DNA component, the fast rate of back-exchange characteristic of nucleic acid hydrogens prevented the application of HDX to recognize the surface of the DBE duplex in direct contact with the DBD. As a possible alternative, we explored the application of transplatin (trans-dichlorodiamineplatinum(II), tPt) to generate protein-DNA cross-links that would help locate the mutual positions of interacting structural features . The spaNon-labelled and isotope-labelled cross-linkers di(N-succinimidyl) glutarate (DSGd0/DSGd4) and di(N-succinimidyl) suberate (DSSd0/DSSd4) were purchased form ProteoChem . Modified protease trypsin  was purchased from Promega . Nuclease Bal-31 was obtained from New England BioLabs . Liquid chromatography solvents of LC/MS grade were purchased from Thermo Fisher Scientific . Other chemicals (highest available purity) were obtained from Sigma-Aldrich. All other chemicals, solvents and buffers for SDS-PAGE were obtained from Bio-Rad Laboratories, Inc. . The pET-15b plasmid carrying His-tag, thrombin cleavage site, and FOXO4-DBD  sequences was obtained from Prof. Obsil.Both, forward and reverse, complementary oligonucleotide strands  were purchased from Integrated DNA Technologies (USA) in standard desalted purity. The reverse oligonucleotide sequence contained DBE (TTG TTT AC) originating from human NOXA promoter localized on chromosome 18 (+ strand) in the position 59,899,552 to 59,899,564 . Both stThe consensus binding sequence of FOXO4 was obtained by searching the HOmo sapiens COmprehensive MOdel COllection (HOCOMOCO) v11 , which c2+. The captured protein was submitted to thrombin digestion to eliminate the tag, followed by gel permeation chromatography. A more detailed description of all experimental procedures is included in the Full-length DBD (residues 82–207 of the entire FOXO4 sequence) was expressed with an N-terminal His-tag from an appropriate pET-15b plasmid, and then affinity captured on a TALON Superflow Resin  charged with Com/z for 3 min  to bridge susceptible amino or hydroxy groups that may be respectively placed within 20.5 ± 3.0 or 24.2 ± 3.0 Å of one another see . The uti147 to either K170 or N-term dropped from 95.9% and 92.8% to 4.1% and 7.2%, respectively , onto the putative binding site of the protein. This operation was accomplished in HADDOCK [The HDX and XL experiments provided a wealth of information that was used to guide the molecular modelling of a full-fledged FOXO4-DBD and FOXO4-DBD•DBE complex. Our approach took advantage of available high-resolution structures that, although incomplete in their coverage of the protein sequence, still represented excellent templates for homology modelling operations , which were somewhat longer in our model , with the DBE component oriented in the proper direction and placed in the correct position onto the FOXO4-DBD’s binding site see . RMSD coodel see . These dodel see . The DBEodel see . Whereasodel see .103–T130, in particular) suggested variations of dynamics upon DBE binding, whereas their subsequent decreasing rates were consistent with the actual structural stabilization resulting from the presence of bound DNA. The RMSD values calculated for corresponding heavy backbone atoms provided an excellent measure of these conformational effects , which were obtained by NMR and crystallography, are not only incomplete, but in the case of crystallography also static. Our extensive data provide a wealth of new information on the conformational dynamics of the protein in both unbound and bound forms. Our experiments clearly differentiated regions that were conformationally stable from those that underwent significant conformational changes upon DNA binding. The most important finding was that binding affected not only the interface region, but also the conformation of regions that were located away from the interface. This information might be essential to understand the allosteric properties of the complex and their role in recruiting additional transcriptional factors.Our models confirmed that full-length FOXO4-DBD adopts the classic forkhead topology characteristic of this family of transcription factors, but corroborated also its unusual DNA binding mode that is unique among those manifested by the highly homologous FOXO proteins . The cloIn conclusion, this study demonstrated the merits of structural proteomics approaches for the elucidation of protein-nucleic acid complexes. The utilization of specific hydrolytic procedures to complete the characterization of HDX and cross-linking products virtually eliminates any limitation pertaining the size of the species of interest. Propelled by continued advances in the computational approaches employed to translate the experimental results into all-atoms models, structural proteomics has rapidly emerged as a valid complement, and often alternative, to the classic high-resolution techniques. By clearly demonstrating the applicability to transcription factor-response element complexes, we hope that this study will lead to a broader utilization of structural proteomics to mitigate the chronic dearth of information on these essential regulatory systems.


Recent researches have suggested that the complex three-dimensional structures caused by structural remodeling play a key role in atrial fibrillation (AF) substrates. Here we aimed to investigate this hypothesis using a multi-layer model representing intramural microstructural features.The proposed multi-layer model was composed of the endocardium, connection wall, and epicardium. In the connection wall, intramural fibrosis was simulated using fibrotic patches randomly scattered in the myocardial tissue of fibrotic layers, while endo-epicardial dissociation was simulated using myocardial patches randomly scattered in the fibrotic tissue of isolation layers. Multiple simulation groups were generated to quantitatively analyze the effects of endo-epicardial dissociation and intramural fibrosis on AF stability, including a stochastic group, interrelated groups, fibrosis-degree-controlled groups, and dissociation-degree-controlled groups.1. Stable intramural re-entries were observed to move along complete re-entrant circuits inside the transmural wall in four of 65 simulations in the stochastic group. 2. About 21 of 23 stable simulations in the stochastic group were distributed in the areas with high endo-epicardial dissociation and intramural fibrosis. 3. The difference between fibrosis-degree-controlled groups and dissociation-degree-controlled groups suggested that some distributions of connection areas may affect AF episodes despite low intramural fibrosis and endo-epicardial dissociation. 4. The overview of tracking phase singularities revealed that endo-epicardial dissociation played a visible role in AF substrates.The complex intramural microstructure is positively correlated with critical components of AF maintenance mechanisms. The occurrence of intramural re-entry further indicates the complexity of AF wave-dynamics. Atrial fibrillation (AF) is a progressive supraventricular arrhythmia characterized by rapid and disorderly atrial electrical activity . For mosin vivo mapping data by quantifying the percentage of fibrillation waves, reporting that the breakthrough incidence and endo-epicardial dissociation degree increased with the complexity of the AF substrate. Gutbrod et al. [ex vivo preparations from diseased hearts, revealing the critical importance of better understanding the role of complex atrial microstructure in AF maintenance.Experimental and clinical studies have suggested that atrial fibrosis might be a mark of structural remodeling in AF –7. Patchd et al.  acquiredd et al.  found thIncreasing evidence suggests that the AF substrate should be more fully described as a 3D complex structure , 15. In According to feasible intramural microstructures derived from the published histological sections of structural remodeling in AF –21, we d3 , , , , and .The dissociation-degree-controlled group: with the same distributions of myocardial blobs in the isolation layers, twelve simulations had the randomized distributions but the same number of fibrotic blobs in the fibrotic layers. The simulation parameters for five groups were , , , , and .It should be noted that intramural fibrosis degree was controlled within a certain range in terms of the relevant quantitative blobs . To ensuThe arrhythmogenic properties of intramural fibrosis and endo-epicardial dissociation were investigated by analyzing the stability and complexity of chaotic electrical signals for all simulations. To effectively reflect the ability to maintain sustained signals, a simulation was evaluated to be able to maintain AF when the electrical signals were stable on the tissue more than 10 s. Therefore, all simulations were classified as stable and non-stable disorders on the basis of their time duration. Complexity was assessed by tracking phase singularities (PSs) of the membrane potential distribution, inspecting electrogram morphologies, and detecting intramural re-entry circuits and breakthrough events.θ at each site t is defined by the following equation:Vmyo(t) is the transmembrane potential at site Vmean is the average of Vmyo during the whole time, and τ is a time window of 30 ms. Phase values lie between −π and π. The location of a PS was detected by the algorithm proposed by Iyer and Gray [t and t+1 ms was less than 1 mm, these two PSs were labelled as the same identification number.A PS is a mark of the tip of a spiral wave. The phase and Gray . Therefoe was computed based on the approximation for a large unbounded volume conductor given by the following equation [Vmyo is the spatial gradient of the transmembrane potential Vmyo; K is a constant associated with the ratio of the extracellular and intracellular conductivity tensors; R is the distance from the source point dv is the differential volume. A bipolar electrogram was then calculated by taking the difference between the two local unipolar electrograms with an interelectrode distance of 6 mm. The simulated ECG was reconstructed by subtracting two unipolar electrograms recovered from points 3 cm above the centers of the endocardium and epicardium sheets. All signals were recorded at 1 kHz and evaluated by Fourier power spectrum analysis.Pseudo-unipolar electrograms at different points of the endocardial and pericardial surfaces were simulated according to uniform intracellular anisotropic resistivity . Therefoequation :Φe(r)=−Breakthrough waves were identified to appear in the endocardium or epicardium sheet by penetrating through any myocardial patch in the isolation layers and being unconnected with the propagation of other waves in the same layer. A veritable breakthrough was required to produce a long enough propagation to affect the propagation of other waves in that layer, rather than being annihilated in a very short time, as shown in P<0.05 was considered as statistically significant.Statistical tests were used to analyze the data and test reasonable hypotheses. For the stochastic group, eight datasets were extracted, including the first 28 and last 28 simulations after ranking based on dissociation degree, the first 28 and last 28 simulations after ranking based on intramural fibrosis degree, the first 20 and last 20 non-stable simulations after ranking based on dissociation degree, and the first 20 and last 20 non-stable simulations after ranking based on intramural fibrosis degree. The Kolmogorov-Smirnov test and independent sample t-test were performed to test the datasets from the stochastic group, the fibrosis-degree-controlled groups, and the dissociation-degree-controlled groups. For all simulations of the stochastic group, three different categories of transmembrane potential maps were presented in P<0.000005 suggested that there was an obvious difference between the results of simulations with high and low endo-epicardial dissociation. Similarly, P<0.0003 suggested an obvious difference between the results of simulations with high and low intramural fibrosis. Significantly simulations with high endo-epicardial dissociation and high intramural fibrosis were more likely to obtain stable episodes of AF.Eight interrelated groups were generated for analysing the effect of the gradual change of intramural microstructure on AF maintenance. A time map of 104 simulations in the interrelated groups is shown in Five fibrosis-degree-controlled groups and five dissociation-degree-controlled groups were used to have a contrast effect. Comparing to the fibrosis-degree-controlled groups, all simulations of each dissociation-degree-controlled group were designed to have the same myocardial blobs in isolation layers. A time distribution, Kaplan-Meier curves, and boxplots for the fibrosis-degree-controlled groups are presented in An overview of phase singularities in the stochastic group is presented in Figs Figs In the present study, we evaluated the role of intramural fibrosis and endo-epicardial dissociation in AF stability by utilizing our multi-layer computational model. Stable intramural re-entries were observed in four simulations. A complex intramural microstructure with high intramural fibrosis and endo-epicardial dissociation could easily maintain AF episodes. In the meantime, there were differences between the effects of intramural fibrosis and endo-epicardial dissociation on the wave-dynamics of AF.Clinical and experimental studies have suggested that AF drivers represent re-entry, ectopic foci, rotor, random wavelets, CFAEs, endo-epicardial breakthroughs, and a complex mixture of all these mechanisms . Stable By combining the simplified simulation of transmural atrial microstructure composed of endo-epicardial dissociation and intramural fibrosis, our proposed multi-layer 3D computational model was used to quantitatively analyze the effect of the complex atrial microstructure on the stability of AF. Stable intramural re-entries were observed after spiral waves disappearances during collisions of breakthroughs in four of 65 simulations in the stochastic group. The disappearance moments of spiral waves in these four stable simulations were respectively 4.3, 9.3, 6.4, and 2.2 s. As shown in The importance of a complex atrial microstructure for determining arrhythmia dynamics was recognized by experimental studies using high-resolution simultaneous endo-epicardial mapping approaches , 29, 30.In this study, four different simulation trial and control groups were applied for contrast research, including a stochastic group, interrelated groups, fibrosis-degree-controlled groups, and dissociation-degree-controlled groups. Lifespan and number of phase singularities for all simulations in the stochastic group were computed to visualize electrical activity during AF. Generally, simulation results of all groups demonstrated that a complex intramural microstructure with high endo-epicardial dissociation and high intramural fibrosis could be significantly beneficial to maintaining the stable episodes of AF. On a micro level, subtle changes of endo-epicardial dissociation and intramural fibrosis could affect the propagation of the action potential wave . The com3 and a 3D slab of 40 × 40 × 3.6 mm3. The simulated ECG is restricted to represent the electrical activity of a piece of atrial tissue in limited time, and thus its waveform is different from the measured ECG with morphological variability and fragmentation during AF. The two isolation layers had the same number of myocardial blobs, and the two fibrotic layers had the same number of fibrotic blobs. These radiuses of cylindrical blobs in isolation and fibrotic layers were set to different constants, and the ionic current properties were spatially uniform in the different types of atrial tissue. These settings need to be further developed in our multi-layer model to approach the complexity of intramural microstructures in human atria. Although different radiuses of cylindrical blobs in fibrotic layers could categorize fibrosis into distinct patterns, our current study was concerned only with patchy fibrosis. Future studies will have to address the difference between the arrhythmogenic potentials of patchy fibrosis and interstitial fibrosis. Additionally, a simulation with the continuous emergence of triggers [Although incorporating a simplified simulation of intramural microstructures, our multi-layer model could not establish a ground truth representation  for atritriggers  due to tHere we describe a novel multi-layer computational model. To our knowledge, this is a more feasible model authentically simulating endo-epicardial dissociation and intramural fibrosis by utilizing different distributions of fibrosis and myocardial patches in a multi-layer 3D slab composed of the epicardium, connection wall, and endocardium. Simulations demonstrated that a complex intramural microstructure with high intramural fibrosis and endo-epicardial dissociation significantly increased the probability of the maintenance of AF episodes, and endo-epicardial dissociation was one of the key factors on the dynamics of electrical activity during AF. The occurrence of intramural re-entry further suggested the importance of adding a complex intramural microstructure to the AF substrate.S1 MovieOne non-stable disorder with the rotor center of spiral wave moving away the endocardium sheet after colliding with breakthroughs.(AVI)Click here for additional data file.S2 MovieOne stable disorder with spiral waves sustaining on the endocardium sheet even with collisions of breakthroughs.(AVI)Click here for additional data file.S3 MovieOne stable disorder maintaining with an intramural re-entry after spiral wave disappearing in the collisions of breakthroughs.(AVI)Click here for additional data file.


There is a great interest in incorporating catechol moieties into polymers in a controlled manner due to their interesting properties, such as the promotion of adhesion, redox activity or bioactivity. One possibility is to incorporate the catechol as end-group in a polymer chain using a functional initiator by means of controlled polymerization strategies. Nevertheless, the instability of catechol moieties under oxygen and basic pH requires tedious protection and deprotection steps to perform the polymerization in a controlled fashion. In the present work, we explore the organocatalyzed synthesis of catechol end-functional, semi-telechelic polylactide (PLLA) using non-protected dopamine, catechol molecule containing a primary amine, as initiator. NMR and SEC-IR results showed that in the presence of a weak organic base such as triethylamine, the ring-opening polymerization (ROP) of lactide takes place in a controlled manner without need of protecting the cathechol units. To further confirm the end-group fidelity the catechol containing PLLA was characterized by Cyclic Voltammetry and MALDI-TOF confirming the absence of side reaction during the polymerization. In order to exploit the potential of catechol moieties, catechol end-group of PLLA was oxidized to quinone and further reacted with aliphatic amines. In addition, we also confirmed the ability of catechol functionalized PLLA to reduce metal ions to metal nanoparticles to obtain well distributed silver nanoparticles. It is expected that this new route of preparing catechol-PLLA polymers without protection will increase the accessibility of catechol containing biodegradable polymers by ROP. Catechol-derivatives and their corresponding ortho-quinones represent one of the most important classes of naturally abundant small molecules due to their capacity to mediate fascinating chemical reactions. Indeed, catechol molecules not only have demonstrated to be potent antioxidant, anti-inflammatory, redox active, or to present outstanding adhesion to different substrates but also they could be easily oxidized. When oxidized, this compound possesses the capacity to react towards a wide variety of chemical groups such as amines or thiols. It is not surprising, that these amazing properties of catechols have opened the door to the design of new (multi)functional polymers based on catechols. Catechols are used in several fields, such as tissue engineering, due to biocompatible and non-toxic properties, for surface coating to protect or improve the adhesion of a given material to a substrate ,2,3,4,5,Catechol functionality can be incorporated into polymers using different strategies. The most studied strategy is the direct polymerization of functional catechols such as dopamine either by oxidative  or enzymN-carboxyanhydrides NCAs containing protected catechols followed by their deprotection 0/[I]0 was varied from 10 to 100 (1H NMR.As seen in 0 to 100 . We foun4 aqueous solution. One oxidation peak is observed in the anodic scan at 0.69 V (A1) and two reduction peaks at 0.43 V (C1) and 0.23 V (C2) at a 5 mV·s−1 scan rate. These redox peaks correspond to the oxidation and reduction processes from catechol to o-quinone via a two-electron two-proton process [The redox behavior of catechol moiety is largely affected by the electrolyte and the presence of protons in the electrolyte solution . Therefo process . When in3/CH3OH solution. The solution changes color indicating that some reactions might have been taking place. To verify the quinone formation, we followed the change in the characteristic signals of dopamine aromatic region by 1H NMR spectroscopy. We found that due the higher electron-withdrawing property of the carbonyl groups there is an effect in the shielding effect on the protons confirming that the catechol was converted to ortho-quinone. 1H NMR spectra of the reaction of oxidized catechol and hexylamine. As soon as the reagents were mixed, the solution turned dark red. The amine can react through two different mechanisms with the o-quinone via the Michael addition or via Schiff base reaction. When the Michael addition is carried out, the amine attach to the quinone in beta position, and via Schiff base an imine group is formed [One of the advantages of catechol units is their ability to be easily oxidized into highly reactive o-quinones allowing its functionalization either by Michael addition or Schiff base reaction. The reaction between catechols and amines is vital in biological processes such as the crosslinking of adhesive proteins by marine organism . In ordes formed ,22. As e0) with small particle sizes were already prepared by adding a silver nitrate (AgNO3) aqueous solution to a catechol containing polymers [+ into Ag0 while the polymer can stabilize the so-formed nanoparticles. Therefore, we explored the utilization of catechol end-functional PLLA for the synthesis of AgNPs using the catechol group as reducing agent and the PLLA with a degree of polymerization of 50 polymer chain as steric stabilizer. For comparative purposes PLLA without catechol units was also investigated as precursor for the synthesis of AgNPs. In this regard, PLLA with a degree of polymerization of 50 was synthesized using phenethylamine as initiator (Catechol is also known to have the ability to reduce metal ions to metal nanoparticles. For instance, elemental AgNPs (Agpolymers . Indeed,nitiator , entry 9nitiator a. A yellnitiator a. The nanitiator a. In thenitiator b.l-lactide using dopamine as initiator is shown. It was demonstrated that the dopamine can be used as initiator for the ring-opening polymerization of cyclic monomers such as l-lactide in the presence of triethylamine as catalyst. The polymers were analyzed by 1H NMR, SEC-IR and MALDI-TOF and the results confirmed the controlled nature of the polymerization and end-group fidelity. We show that this catechol end-functional PLLA polymer can be easily converted to highly reactive ortho-quinone using 3-Dichloro-5,6-dicyano-1,4-benzoquinone oxidizer. After the oxidation step, we confirmed that the catechol-PLLA polymer can be post-functionalized with amines. Besides the ability of catechol to be post-functionalized, we also confirmed the ability of catechol functionalized PLLA to reduce metal ions to metal nanoparticles, studying the formation of silver nanoparticles. It is expected that this new route of preparing catechol-PLLA polymers without protection will increase the accessibility of catechol containing biodegradable polymers by ROP.In this work, the controlled organocatalyzed ROP of


In the present work, a soft lithographic process is used to create nanometer-sized line patterns of gold nanoparticles (Au NPs) on PEG-based hydrogels. Hereby nanometer-sized wrinkles on polydimethylsiloxane (PDMS) are first fabricated, then functionalized with amino-silane and subsequently coated with Au NPs. The Au NPs are electrostatically bound to the surface of the wrinkled PDMS. In the next step, these relatively loosely bound Au NPs are transferred to PEG based hydrogels by simple contacting, which we denote “nano-contact transfer”. Nano-patterned Au NPs lines on PEG hydrogels are thus achieved, which are of interesting potential in nano-photonics, biosensor applications (using SERS) and to control nanoscopic cell adhesion events. Gold nanoparticles (Au NPs) have many interesting and useful properties such as low biological toxicity, conductivity, easy functionalizability, and size- and shape-dependent localized surface plasmons, which can be tuned towards several applications ranging from inorganic electronic devices to spectroscopy, biosensors and biological applications . Among oHydrogels are three-dimensional networks of hydrophilic polymers, which absorb large amounts of water without losing the network structure and without being dissolved in the aqueous solution, similar to organic tissues . In ordeEspecially PEG-based hydrogels are suitable due to their non-toxicity, non-immunogenicity and non-fouling characteristics that offer excellent background properties to study fundamental cellular processes, such as adhesion, migration and proliferation . HydrogeNotwithstanding their great use and potential, these microfabrication techniques inherently produce micrometer-sized features. As mentioned before, in many cases, control of patterns at the nanometer scale is necessary as well. This can be achieved by nanofabrication methods, e.g., electron beam lithography (EBL) , dip-penAs mentioned above, Spatz et al. presented different possibilities for patterning Au NPs on different (hard) substrates at nano- and micrometer scales using the block copolymer micelle nanolithography. Further, for patterning of hydrogels, Ding et al. developed a process where the patterned Au NPs on glass could be transferred onto the surface of hydrogels . Hereby Not only for cell culture but also for spectroscopic devices; biosensors or nanophotonic materials, precise control over Au NP patterns at the nanometer scale is necessary. To that end, Fery et al. ,28 used In this work, we present a process to create controlled nanometer-sized patterns of Au NPs on PEG based hydrogels. The nano-patterns of Au NPs were achieved by the controlled targeting of the surface-wrinkle sizes of PDMS by tuning the plasma treatment time of the stretched PDMS according to the principles introduced by Whitesides et al.  Subseque4·3H2O), trisodium citrate (Na3C6H5O7), poly(ethylene glycol) diacrylate  and 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (photoinitiator-PI Irgacure 2959) were purchased from Sigma-Aldrich Chemie GmbH . (3-Aminopropyl)trimethoxysilane (APTMS) was bought from ABCR GmbH & Co. KG . Poly(dimethylsiloxane) (PDMS) and the curing agent are from Dow Corning GmbH . All the chemicals were used without further purification.Tetrachloroaurate trihydrate . In order to avoid bubbles the mixture was degassed in a desiccator, then casted on flat glass slide and cured 2 h at 120 °C. After the PDMS was cooled down to room temperature, small pieces of PDMS were cut and placed in a stretch apparatus and stretched from one side to 125% of the original sizes of the PDMS pieces. The PDMS were treated with an oxygen plasma  for a predefined period of time in the stretched condition. Subsequently after plasma treatment the PDMS pieces were relaxed. During the relaxation process well-arranged nanometer sized wrinkles on the PDMS surfaces were created.Wrinkled PDMS structures were done respectively to the procedure of Whitesides et al. . FirstlySubsequently after the PDMS wrinkles were created via plasma treatment, the PDMS-stamps were placed in a desiccator containing two drops of amino-silane agent. Then vacuum was kept and left for 2 h.Mw = 575 g/mol) was used as precursor and was mixed with 1% of photoinitiator Irgacure 2959. For having a good distribution of the photo-initiator the mixture was sonicated for around 5 min. Then the mixture was dispensed on a glass slide and covered with a thin glass cover slip to achieve a flat hydrogel sample. This liquid mixture was put under UV-light source for 8 min and the glass cover slip was peeled off. The result was a thin, flat hydrogel sample, as a stand-alone film.PEG hydrogels were prepared using the UV-curing process see . For tha−1·cm−1 [The wrinkled and amino-silanized PDMS was coated with the respectively synthesized Au NPs, which had a concentration of 3.0 nM according to the extinction coefficient of ε = (8.78 ± 0.06) × 108 M−1·cm−1 . Hereby First of all, wrinkled PDMS-stamps were prepared via plasma oxidation of smooth PDMS molds in a stretched configuration, and subsequent relaxation of the stamp to the original macroscopic dimensions . By thisIn the next step, the surfaces of the wrinkled PDMS-stamps were silanized with amino functionalities . For comparison, AFM measurements of the non-silanized PDMS-stamp were performed for the 2 min plasma-treated stamp and no significant different was observed on height images before and after amino-silanization . This siThe sizes of the wrinkles or nano-topographies were controlled by the plasma oxidation time; in general it can be said that the longer the plasma oxidation time the wider the distances of the line spaces and the deeper the grooves were. The distances of the nano-lines (from hill to hill) varied from around 300 up to around 600 nm by choosing a plasma oxidation time of 2 up to 15 min, which can be seen in the AFM images in The amino-silanized surfaces were subsequently coated with Au NPs see . For tha2. The distances of the particles can be further tuned with dilution processes of the Au NPs solutions before coating on the stamp.The distribution of the Au NPs on the surface of the PDMS-stamp is shown in the AFM and SEM images in After having an Au NPs-coated PDMS-stamp, the Au NPs can be transferred via stamping on different hard surfaces like on Si-wafers or glass, or on softer surfaces like on hydrogels. For the nano-contact printing process, the surface properties of both the stamp and the substrate are crucial in determining the transfer efficiency. The molecules or the particles, which will be transferred, should interact with the stamp with an intermediate affinity; the interaction should be strong enough so that the particles can distribute over and cover the stamp properly but also loose enough to be released after contacting with the surface of the eventual goal substrates. In the present work, the interaction of Au NPs with the PEG hydrogels surfaces was likewise important. Here, the Au NPs were stabilized with citrate molecules that provide the Au NPs with a negative surface charge. The amino-groups on the surface of the PDMS-stamp manifest positive charges in aqueous medium, so there were attractive electrostatic interactions.Au NPs were then transferred onto non-reactive, non-charged hydrogels . It can As far as the transfer efficiency is concerned, taking a closer look at the Au NPs-coated PDMS-stamp after contact with the PEG hydrogel see  it couldIn order to improve the transfer efficiency, softer PEG hydrogels could be used, which will have a better conformal contact with the stamp surface and peel off the Au NPs more efficiently. Besides the simple physico-chemical properties, also chemical interactions of the hydrogel can be further tuned. The substrate is needed to be more attractive towards the Au NPs than this electrostatic interaction in order to take up the Au NPs from the stamp efficiently. In that case mercapto-functionalized hydrogel substrates will be an option, due to the strong covalent binding affinity of the Au NPs with thiol-functions. Currently, we are exploiting reactive, thiolated hydrogels that have great affinity to Au NPs .Another possibility to increase the transfer efficiency is to cover the surface with the liquid PEG–prepolymer and do the UV-crosslinking directly on the surface of the Au NPs-patterned stamp and peel off the cured hydrogel from the surface by swelling according to our other contribution in this special issue . This woSuch Au NPs-patterned PEG–hydrogels are also of great interest to control cell adhesion with nanoscopic precision, as we have recently reported ,26,38. IIn this work we presented an easy and fast soft-lithographic method to achieve nano-sized patterns of Au NPs with different sizes on PEG hydrogels without utilizing any specifically prefabricated silicon masters, but rather using only soft, polymeric materials as molds, stamps and substrates.The pattern sizes were originally controlled via the wrinkled PDMS surface structure, while tuning of the plasma treatment times. In general, it could be stated that the longer the plasma treatment time, the larger are the distances between the grooves of the wrinkles and the deeper are the grooves on PDMS-stamps.In the second step, the wrinkled PDMS was surface-functionalized with amino-silane molecules and coated with citrate-capped Au NPs. Thus, the Au NPs were bound on the surface of the PDMS via effective, yet non-covalent, electrostatic interactions.In parallel, smooth and flat films of PEG-based hydrogels were prepared to serve as the versatile and inert, biomaterial goal substrate.The nano-patterns of Au NPs were subsequently transferred onto the surface of the PEG–hydrogels through contacting; a process that we denote “Nano-Contact Transfer”.Especially the non-toxicity and non-fouling characteristics of PEG based materials and the size-and shape-dependent optical properties and the low toxicity and make these materials highly useful for biological or biomedical devices, tissue engineering, drug delivery and cell biological studies. Other applications are localized surface plasmon resonance (LSPR) or surface enhanced Raman spectroscopies (SERS), where specifically designed Au NPs areas on surfaces are indispensable because the surface plasmons of the Au NPs are affected by the distances among the particles by the overlapping of plasmon states of neighboring particles.