The hierarchical modular organization of functional networks in the brain is crucial for supporting diverse cognitive functions. Functional disorders in the brain are associated with an abnormal hierarchical modular organization. The default mode network (DMN) is a complex dynamic network that is linked to specialized cognitive functions and clinically relevant information. In this study, we hypothesize that hierarchical functional segregation and integration of the DMN within attention-deficit/hyperactivity disorder (ADHD) is abnormal. We investigated topological metrics of both segregation and integration in different hierarchical subnetworks of the DMN between patients with ADHD and healthy controls. We found that the hierarchical functional integration and segregation of the DMN decreased and increased, respectively, in ADHD. Our results also indicated that the abnormalities in the DMN are intrinsically caused by changes in functional segregation and integration in its higher-level subnetworks. To better understand the temporally dynamic changes in the hierarchical functional integration and segregation of the DMN within ADHD, we further analyzed the dynamic transitions between functional segregation and integration. We found that the adaptive reorganizational ability of brain network states decreased in ADHD patients, which indicated less adaptive regulation between the DMN subnetworks in ADHD for supporting correspondingly normal cognitive function. From the perspective of hierarchical functional segregation and integration, our results further provide evidence to support dysfunctional brain cognitive functions within ADHD linked to brain network segregation and integration. Attention-deficit/hyperactivity disorder (ADHD) is one of the most common and severe impairments of psychological development in children, with a prevalence of 0.1–8.1% worldwide; further, approximately 50% of patients are affected until adulthood . HoweverMany studies have shown that ADHD is caused by abnormal communication within functional networks of the brain , 4. BraiRecently, several studies have suggested that brain networks possess a hierarchical modular organization , 15, whiPrevious studies have suggested that an abnormal default mode network (DMN) plays a critical role in mental disorders, such as depression and ADHD , 18–20. In this study, we hypothesize that the hierarchical functional segregation and integration of the DMN within ADHD is abnormal. We investigate the difference in segregation and integration between patients with ADHD and healthy controls by analyzing topological metrics in the hierarchical subnetworks of the DMN topological metrics. In addition, we compare the dynamic transitions between segregated and integrated processing in the hierarchical DMN in ADHD and control subjects.http://fcon_1000.projects.nitrc.org/indi/CoRR/html/ipcas_1.html.) by Milham and Castellanos in December, 2009. These data were acquired from 24 ADHD patients and 24 healthy subjects (control subjects) in the resting state by a 3T Siemens scanner. The ADHD patients in this dataset were evaluated with the Clinical Interview for DSM-IV (SCID), the Symptom Checklist-90-Revised (SCL-90-R) and the Adult ADHD Clinical Diagnostic Scale (ACDS). It should be noted that we used pwr.t.test, a function in R statistical software, to calculate the power in the present analysis with the following parameters: number of samples in each group = 24, large effect size = 0.8, significance level = 0.05, power of test = 0.86. In general, the sample size is acceptable if the testing power calculated is more than 0.8. Image scans contained 39 slices and 192 time points with TR = 2 s, TE = 25 ms, flip angle = 90°, matrix = 64 × 64, FOV = 192 mm2, voxel size = 3 × 3 × 3 mm3, and a final time of 390 s.The resting-state fMRI data used in this research were released to the open-access “1000 Functional Connectomes Project” (http://afni.nimh.nih.gov/afni/) and FSL (http://www.fmrib.ox.ac.uk/fsl/) according to standard preprocessing protocols. First, the first four volumes were removed from the analysis to ensure the initial stabilization of the fMRI signal. Then, through a 3D image realignment with the AFNI program 3dvolreg function, motion correction was carried out for each subject. Images acquired from echo-planar imaging (EPI) were motion and slice-timing corrected, and spatially smoothed using a Gaussian kernel of 6 mm full width at half maximum (FWHM). To minimize the effects of low-frequency drift and high-frequency physiological noise, temporal bandpass filtering (0.01 Hz < f < 0.08 Hz) was performed. As the global component of the fMRI fluctuations measured during the resting state is tightly coupled with the underlying neural activity [The fMRI data were preprocessed using AFNI  with the AAL  atlas , resultist level contains all DMN regions. The 2nd level consists of two subnetworks within each hemisphere of the DMN, i.e., the left DMN (lDMN) and the right DMN (rDMN). Each hemisphere of the DMN is divided into two subnetworks in the 3rd level, i.e., the right anterior DMN (raDMN), the right posterior DMN (rpDMN), the left anterior DMN (laDMN) and the left posterior DMN (lpDMN).The asymmetries of the human brain, in terms of structure and function, have been well-documented and are involved in a variety of functions, such as language, motor, and visuospatial processing . Studiesn is the number of nodes, N is the set of all nodes in the network, ti is the number of edges between the neighbors of node i, and ki is the degree of node i, which represents that node’s number of neighbors. The clustering coefficient quantifies the number of connections that exist between the nearest neighbors of a node as a proportion of the maximum number of possible connections. Thus, a higher clustering coefficient implies a higher extent of segregation in a network. The shortest path length of a network is:The clustering coefficient and shortest path length from graph theory were used to characterize the capability of functional segregation and integration in brain functional networks . The cludij is the shortest path length between nodes i and j of the network, which is an inverse of the connection weight between nodes i and j in a weighted correlation network. Note that dij = ∞ for all disconnected pairs i and j. Shorter paths imply a stronger potential for integration in a network.Here, κis,t is the strength of the positive edge weights of node i to the nodes in module s at time t, ki,t is the strength of the positive edge weights of node i to all the other nodes at time t, and NM is the number of modules. A high value of Pi,t indicates that a node is connected to other nodes in most of the modules in a network, and low values of Pi,t indicate that a node is only connected to other nodes in a single or a small number of modules. A high mean Pi,t over nodes can be associated with the existence of highly integrative processes across the whole network. Here, all calculations in our network analyses are based on the Brain Connectivity Toolbox  in MATLAB.We also adopted the participation coefficient to estimate the segregated and integrated network states. The participation coefficient expresses the level at which a node is coupled with other nodes across all modules and is described as:p < 0.05; exact p values are listed in p > 0.05). We also found an increased shortest path length in the raDMN, rpDMN, laDMN and lpDMN in ADHD patients. By performing the statistical two-sample t-test, we confirmed that the changes in the shortest path length caused by ADHD were significant for the lpDMN and rpDMN (p < 0.05) but not for the laDMN and raDMN (p > 0.05).The highest-level of the DMN contains four subnetworks, i.e., raDMN, rpDMN, laDMN and lpDMN . To invend level, which contains the lDMN and the rDMN. Structurally, the lpDMN and laDMN are integrated into the lDMN, and thus, we first focused on the integration between these two modules. As shown in p < 0.05), indicating a stronger functional integration between the lpDMN and the laDMN. Combining the decreased functional integration and increased segregation in the laDMN and lpDMN caused by ADHD, it is interesting to further investigate how the functional properties change in the lDMN. This result is shown in p < 0.05). Meanwhile, the shortest path length of the lDMN was significantly increased in the ADHD patients , indicating decreased functional integration and increased functional segregation in the lDMN caused by ADHD.We further investigated how the functional changes at the highest level were integrated to form abnormal segregation and integration at the 2p > 0.05), indicating that ADHD has an insignificant effect on the integration between the rpDMN and raDMN. We then calculated the clustering coefficient and the shortest path length for the rDMN. As shown in p < 0.05 for both the clustering coefficient and the shortest path length). Thus, ADHD induces decreased functional integration and increased segregation in the rDMN by affecting the functional properties in the highest-level subnetworks. Apparently, the lDMN and rDMN can be integrated into the whole DMN. p < 0.05), indicating stronger integration between the lDMN and rDMN. This is similar to the result for the lDMN. Furthermore, the clustering coefficient and the shortest path length for the DMN are significantly higher in the ADHD patients than in the control subjects (p < 0.05), indicating decreased functional integration and increased segregation. Here, our results reveal that abnormalities in the DMN are intrinsically aroused by the changes in functional segregation and integration in the higher-level hierarchies.Meanwhile, the rpDMN and raDMN are structurally integrated into the rDMN. As shown in k-means clustering algorithm (k = 2). The cluster with higher participation coefficients on average is regarded as the cluster of the integrated state. As shown in Brain functional networks dynamically switch between segregated and integrated processing, and these flexible transitions consist of specific patterns that are dependent on brain state –40. To ip > 0.05). We further investigated the proportion of the integration state over time for subnetworks in the 2nd level. Interestingly, we found that the mean values of the proportion of the integration state over time for the lDMN and rDMN in the ADHD patients were higher than those in the control subjects, and the change in the rDMN was statistically significant . Note that this is contrary to the results from the highest-level networks. At the 1st level, the mean values of the proportion of the integration state over time for the DMN also improved under the influence of ADHD (p > 0.05).To investigate the dynamic integration ability, we calculated the proportion of the integration state over time for different subnetworks . The meap < 0.05). As integrated to lDMN and rDMN, the number of flexible transitions was also reduced in the ADHD patients, and this change was significant for lDMN  but not for rDMN (p > 0.05). As mentioned before, the lDMN and rDMN are integrated into the whole DMN. Significantly fewer transitions between the functional segregation and integration states were performed in the DMN for the ADHD patients (p < 0.05).Flexible transitions between functional segregation and integration are crucial for normal brain cognitive functions. To investigate the effect of ADHD on brain flexibility across multiple levels of the DMN, we calculated the frequency of the transition between functional segregation and integration in the time series for each subnetwork . The fouIn this study, we investigated functional segregation and integration features between ADHD patients and control subjects across hierarchical subnetworks of the DMN system. We found that abnormalities within the subnetworks of the DMN in ADHD are heterogeneous, and a dysfunctional DMN is essentially caused by changes in functional segregation and integration of its higher-level subnetworks. More interestingly, through the analysis of hierarchical dynamic transitions, we also found that the states of functional segregation and integration in the ADHD patients are more stable, which means that there is less adaptive regulation between the DMN subnetworks in the ADHD patients.st level contains all DMN regions, the 2nd level includes the lDMN and rDMN, and the 3rd level consists of the raDMN, rpDMN, laDMN and lpDMN.We investigated the difference in the functional segregation and integration of the hierarchical DMN between the ADHD patients and the control subjects. We used a method to provide a hierarchical modular organization, which has a strong correspondence between the structure and functions of the DMN , 34. In rd level of the DMN, the clustering coefficient and the shortest path length of the lpDMN and rpDMN in the ADHD patients are significantly higher than in the control subjects, but these network metrics are not significant for the laDMN and raDMN. In the 2nd level of the DMN , the functional connectivity between the lpDMN and laDMN is significantly increased in ADHD patients, but it is not significantly different between the rpDMN and raDMN. More importantly, the clustering coefficient and shortest path length for both the lDMN and rDMN in the ADHD patients were statistically higher than those in the control subjects. It is further observed that the functional connectivity between the lDMN and rDMN is significantly increased in the ADHD patients, and the clustering coefficient and shortest path length for the DMN in the ADHD patients are statistically higher than those in the control subjects.The DMN is associated with attention-related brain cognitive functions. A dysfunctional DMN is often linked to attention deficits and mental diseases, such as depression and ADHD –20. Our A higher clustering coefficient indicates that there are multiple communities in a subnetwork, which means that the functional segregation capability is increased in ADHD patients. Higher shortest path length means that the ability to process information is decreased in ADHD patients, and the functional integration capability is weakened. Many previous studies have indicated increased functional segregations and decreased functional integrations in the brain networks of ADHD patients , 13, 41.Recently, studies have demonstrated the functional heterogeneity of the different brain regions in the DMN , 43. EacMore interestingly, the lDMN and rDMN are regularly integrated by the highest-level subnetworks , and both show functional segregation hyperactivity and a decrease in functional integration capability. These results may indicate that the functional change in the posterior DMN plays a key role in ADHD. The overall network characteristics of the DMN are determined by different subnetworks. These findings may enhance our understanding of the dysfunction in the DMN in ADHD patients.Furthermore, the interaction of the subnetworks within the DMN also makes some important contributions to the construction of brain network segregation and integration. It should be noted that functional connectivity represents the capability of brain areas/networks to interact with information. The increased functional connectivity of the laDMN-lpDMN and the lDMN-rDMN may suggest an increase in local efficiency (increase in network segregation) of hyperactivity information processing in ADHD. Indeed, a decrease in global brain efficiency (decrease in network integration) and an increase in local brain efficiency have been confirmed in juvenile ADHD patients and adults , 48. Abnrd level subnetworks in the ADHD patients, the proportion of the integration state was lower than that in the control subjects. However, for subnetworks in the 2nd level and 1st level in the ADHD patients, the proportion is higher than that in the control subjects   . The morThe notion that brain dynamics work on a critical point is growing in support . At thatThere were a number of limitations in our study. First, our current study is limited by the small sample size. Although we have evaluated the power result, the sample size should be increased in future work. Second, the data we used here was acquired from resting state fMRI, and future studies should include data from task-state fMRI to study the cognitive competence of ADHD. Third, the present study focused on the hierarchical DMN. More work and investigations on the other hierarchical brain functional networks or the whole brain functional network will be included in the future.In this study, we investigated both segregation and integration brain network topological metrics between ADHD and healthy controls. We provided new insights to explain the brain cognitive dysfunction of ADHD from the view of hierarchical functional segregation and integration. We found that abnormalities between subnetworks in the DMN in ADHD patients are heterogeneous, and DMN dysfunction is essentially caused by changes in functional segregation and integration of its higher-level subnetworks. In addition, through the analysis of hierarchical dynamic transitions, we also found that the states of functional segregation and integration in ADHD patients are more stable, which means that there is less adaptive regulation between DMN subnetworks in ADHD patients. Our results can help us better understand the abnormal brain network mechanism of ADHD and serve as a clinical biomarker of ADHD.


Increasing attention is payed to the contribution of somatosensory processing in motor control. In particular, temporal somatosensory discrimination has been found to be altered differentially in common movement disorders. To date, there have only been speculations as to how impaired temporal discrimination and clinical motor signs may relate to each other. Prior to disentangling this relationship, potential confounders of temporal discrimination, in particular age and peripheral nerve conduction, should be assessed, and a quantifiable measure of proprioceptive performance should be established.To assess the influence of age and polyneuropathy (PNP) on somatosensory temporal discrimination threshold (STDT), temporal discrimination movement threshold (TDMT), and behavioral measures of proprioception of upper and lower limbs.STDT and TDMT were assessed in 79 subjects . STDT was tested with surface electrodes over the thenar or dorsal foot region. TDMT was probed with needle electrodes in flexor carpi radialis (FCR) and tibialis anterior (TA) muscle. Goniometer-based devices were used to assess limb proprioception during (i) active pointing to LED markers, (ii) active movements in response to variable visual cues, and (iii) estimation of limb position following passive movements. Pointing (or estimation) error was taken as a measure of proprioceptive performance.In healthy subjects, higher age was associated with higher STDT and TDMT at upper and lower extremities, while age did not correlate with proprioceptive performance. Patients with PNP showed higher STDT and TDMT values and decreased proprioceptive performance in active pointing tasks compared to matched healthy subjects. As an additional finding, there was a significant correlation between performance in active pointing tasks and temporal discrimination thresholds.Given their notable impact on measures of temporal discrimination, age and peripheral nerve conduction need to be accounted for if STDT and TDMT are applied in patients with movement disorders. As a side observation, the correlation between measures of proprioception and temporal discrimination may prompt further studies on the presumptive link between these two domains. Temporal aspects of somatosensory processing have drawn increasing interest as potential markers in the differential diagnostic workup of movement disorders. In particular, STDT and TDMT have been shown to be differentially involved. STDT is a neurophysiological paradigm testing the shortest ISI at which a subject can perceive successive electrical stimuli applied to the skin as separate. Higher STDTs have been consistently described in several types of dystonia , 2008 asTo date, there have only been speculations as to how an impairment of temporal discrimination performance and clinical motor signs in these movement disorders may relate to each other , warrantTo this end, we assessed STDT and TDMT of upper and lower extremities in healthy subjects of different age and in patients with PNP, combined with a set of three proprioception tasks of the corresponding limbs. We hypothesized that higher age and PNP are associated with (i) increased discrimination thresholds and (ii) decreased proprioceptive performance.The study conformed to the principles of the Declaration of Helsinki. It was approved by the Ethics committee of the Medical Faculty at the University of Würzburg.A total of 54 volunteers without a history of neurological or psychiatric disease and without clinical symptoms or signs of such disease were included. Additional exclusion criteria were a medical history of diabetes or coagulation disorders, and ongoing medication with oral anticoagulant drugs. In addition, 25 patients with a diagnosis of chronic PNP were included. Nerve conduction studies and SSEPs were collected in order to characterize PNP patients, and to exclude impairment of peripheral nerve or posterior column conduction in healthy subjects older than 60 years. Conduction studies were performed at our Clinical Neurophysiology Laboratory  according to the clinical standard.All participants gave their written informed consent for research.Somatosensory temporal discrimination threshold was tested with surface electrodes (anode and cathode with 1 mm diameter and 1.5 cm distance in between) placed over the thenar or dorsal foot region. Pairs of square wave electric stimuli with a duration of 0.2 ms were provided by a constant current stimulator . Stimulation intensity was determined individually by providing stimuli with stepwise increasing current until participants were able to perceive stimuli clearly . ISIs were presented in an ascending sequence, starting from 0 ms, in steps of 5 ms. STDT was defined as the shortest ISI when participants perceived two separated pulses in three successive intervals , 2013a. TDMT was measured following the procedure described by The mean value of four runs was taken for further analysis.Proprioception of limbs was assessed by custom-made goniometers without visual feedback of the respective extremity throughout the testing procedure . The devSubjects were instructed to perform three different proprioceptive tasks as precisely as possible, and to initiate limb movements only on explicit request by the investigator. The first task (LED) comprised pointing to LED markers attached to the outside of the device at 15°, 30°, 45°, 60°, and 75° for the upper limbs , and 15°U test for group comparisons and the Spearman test for correlations. Statistical significance was set at a level of p < 0.05. The Benjamini–Hochberg procedure was used to correct for multiple comparisons.SPSS software (IBM) was used for statistical analyses. We tested for normality by using the Shapiro–Wilk test. As data were not normally distributed, we applied the Mann–Whitney A total of 54 healthy subjects  with a median age of 54 (range 30–76) years were included into this study. In addition, 25 patients  diagnosed with PNP with a median age of 61 (range 46–79) years were included. Demographic and clinical data of PNP patients and a subgroup of age- and sex-matched healthy controls are summarized in The results of STDT and TDMT assessment are presented in the upper part of r = 0.348; p < 0.001; r = 0.581; p < 0.001; r = 0.267; p = 0.005; r = 0.465; p < 0.001; In the group of healthy controls, higher age was associated with higher discrimination threshold levels for STDT of the upper extremities  are presented in the lower part of In the whole group of healthy controls there was no significant correlation between age and higher pointing errors in all performed tasks.Compared to the group of matched healthy participants, patients with PNP performed worse in the pointing tasks , with higher pointing errors for the upper and lower limbs. In the PASSIVE condition, performance was comparable between patients and controls .r = 0.477; p < 0.001; r = 0.546; p < 0.001; r = 0.336; p = 0.002; r = 0.523; p < 0.001; r = 0.369; p = 0.001; r = 0.435; p < 0.001; r = 0.372; p = 0.001; r = 0.460; p < 0.001; Screening the entire data for a potential correlation between the two domains, we found higher temporal discrimination thresholds to be associated with higher pointing errors in the ARROW task at upper  and proprioceptive performance in a group of healthy controls and PNP patients. Our main goal was to determine the influence of age and peripheral nerve conduction on both measures as groundwork for their use in patients with movement disorders. In line with our first hypothesis, higher age and PNP were associated with increased discrimination thresholds. Our second hypothesis, in contrast, was only partially confirmed: PNP, but not age, was associated with decreased proprioceptive performance.Across healthy subjects of different age groups, we found higher age to be correlated with increased temporal discrimination values. In line with a previous study , we founOur finding of significantly increased STDT and TDMT at the upper and lower limbs in patients with PNP points to a considerable impact of peripheral nerve conduction on temporal discrimination. Indeed, blurred signal conduction due to temporal dispersion within the peripheral nerve might well explain our observation of poor temporal discrimination in patients with PNP. As a consequence, peripheral conditions with high general prevalence, like PNP, carpal tunnel syndrome, or radicular compression, might inevitably limit the individual validity of temporal discrimination thresholds in subjects with movement disorders and additional peripheral conditions.As a potential limitation, the group of PNP patients was rather heterogenous with respect to type and severity of neuropathy. While an increase of STDT and TDMT in demyelinating PNP might be more suggestive than in axonal PNP or small fiber neuropathy, the respective impact of different types of nerve damage remains speculative. Moreover, nerve conduction studies predominantly reveal information about the distal part of the nerve, whereas proximal demyelination may be missed easily. Consequently, the attempt to disentangle the causal relationship between a particular finding in nerve conduction studies and temporal discrimination performance would be an overinterpretation of our data. This is even more so since the CNS would not remain unaffected by peripheral neuropathy – plastic processes like cortical rearrangement following PNP-related partial denervation might also affect temporal discrimination on the CNS level.differential association of STDT and TDMT with age or PNP: both parameters changed concordantly, i.e., they were positively correlated with age, and both were similarly increased in PNP patients as compared to healthy subjects. Thus, physiological aging as well as PNP influence STDT and TDMT similarly. This is in line with the observation that specific patterns of altered temporal discrimination in disorders like essential tremor or dystonia can be attributed to specific changes at the level of the CNS. Several brain areas have been suggested by fMRI to be involved in temporal discrimination, first of all the basal ganglia (Notably, we did not find a  ganglia , in part ganglia , and the ganglia . In addi ganglia .We did not find a significant correlation between age and pointing or estimation errors, respectively. Earlier studies have demonstrated an age-related decline of passive finger  and anklPatients with PNP showed an impaired kinesthetic performance as evidenced by higher pointing errors compared to controls. In the light of reduced peripheral afferent input due to PNP, this finding is not surprising . As for As an additional observation, we found correlations between STDT and TDMT and the performance in two different active pointing tasks: Higher temporal discrimination thresholds were associated with higher pointing errors in the ARROW and LED task in upper and lower extremities. Though postulated by earlier studies, which had found differential alterations of STDT and TDMT in patients with movement disorders , the assActive pointing movements rely on permanent “on-line” adjustments of their temporospatial properties in order to achieve a high level of precision . TemporaAs correlations do not allow directional or causal inference, an alternative explanation might be that both temporal discrimination and pointing performance are modulated comparably by a common third parameter. This could either be an unidentified parameter or simply peripheral nerve conduction with PNP-related variability. In the latter case we would expect a lack of correlation when PNP patients are excluded. However, there remained to be a significant correlation between STDT and TDMT and ARROW task in upper limbs in the group of healthy subjects, which supports an association of the two parameters above their common modulation by peripheral conduction.Functional imaging studies on proprioception during either vibration induced illusory motion or passive extremity movements revealed involvement of a number of brain regions partially overlapping with those discussed in the context of temporal discrimination . It is bAge and PNP have significant impact on measures of temporal discrimination and/or proprioceptive capacity. STDT and TDMT increase with age, and PNP is associated with higher STDT/TDMT values and reduced precision in pointing tasks. If applied in studies on movement disorder, where STDT/TDMT may be used in order to define corresponding endophenotypes, it is important to account for these factors to increase validity of the measurements. This is particularly important in view of a significantly higher prevalence of PNP in patients with PD as compared to controls . As an aAll datasets generated for this study are included in the article/The studies involving human participants were reviewed and approved by Ethik-Kommission der Universität Würzburg. The patients/participants provided their written informed consent to participate in this study.TO and DZ designed the study concept and wrote the manuscript with the help of TW. TW and TO carried out the experiments and performed the statistical data analysis.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.


Attention deficit/hyperactivity disorder (ADHD) has been linked to higher engagement in sexual risk-taking behavior (SRTB). The current study aims to establish the link between ADHD symptoms and SRTB in the general population and to examine whether an exaggerated perceived benefit of the positive outcomes of SRTB explains that link. A scale for measuring the frequency, likelihood, perceived benefit, and perceived risk of SRTB was developed. Young adult sexually active participants who did not have a stable partnership completed the above scale, as well as a scale of ADHD symptoms. The level of ADHD symptoms positively correlated with the frequency and likelihood of SRTB, even when the overall level of sexual behavior was controlled for. ADHD symptoms also correlated with the perceived benefit of SRTB, but not with the perceived risk of SRTB. Mediation analysis confirmed an indirect pathway: ADHD symptoms predicted perceived benefit of SRTB, which in turn predicted increased likelihood to engage in SRTB. These findings suggest a positive link between ADHD symptoms and SRTB in the general population, which is accounted for by an exaggerated perceived benefit of SRTB. Attention deficit/hyperactivity disorder (ADHD) is a neurodevelopmental disorder characterized by inattention and/or hyperactivity/impulsivity and is associated with poor academic, social, occupational, financial, and health-related functioning . People Sexual risk-taking behavior may result in a variety of undesirable outcomes, including family conflicts, financial loss, and damaged reputation, but the two most commonly addressed concerns are unintended pregnancies and sexually transmitted diseases, such as HIV/AIDS . SRTB inA small number of studies have focused on the link between ADHD and SRTB. Prospective studies showed that childhood ADHD is associated with earlier sexual activity, a higher number of sexual partners, more sex outside of a relationship, more sexually transmitted diseases, more partner pregnancies, and teenage parenthood .A relevant question is what may explain the link between ADHD and SRTB. Individual differences associated with both ADHD and SRTB may have the potential to act as mediators. Lifetime conduct disorder symptoms, which are closely related to ADHD, were found to contribute to the link between ADHD and SRTB significantly . In anotExcessive SRTB may also result from higher engagement in sexual behavior in general, as extra sexual activity, when not in the context of a stable, monogamous relationship, increases the possibility of exposure to risky sexual intercourse. In the literature regarding ADHD and risk taking in driving, the measured level is usually controlled against the total exposure/driving time of the participant . HoweverThe present study suggests observing SRTB through the prism of Weber’s behavioral decision theory . AccordiTherefore, the goal of the study presented here was to examine the association between ADHD and SRTB and the influence of the perceived benefit on this association. In order to measure SRTB, a questionnaire consisting of 15 risky sexual behaviors was assembled. For each behavior, the participants were asked to rate the frequency, likelihood, perceived benefit, and perceived risk of engagement in that behavior. The frequency section contained an additional item probing for the frequency of engagement in “sexual activity of any kind” (not only risky) to control for individual differences in general sexual activity.In this paper, we favored a dimensional approach for the conceptualization of ADHD and, thus, referred to ADHD as a continuous trait, based on mental health dimensional models . AccordiHence, the present study hypotheses were as follows: (1) ADHD symptoms in the general population would be positively associated with the engagement in SRTB, even after controlling for general sexual activity; (2) an indirect link between ADHD and SRTB would be found through the link between ADHD and benefit perception.The experiment was approved by the ethics committee of the Seymour Fox School of Education, at the Hebrew University. One-hundred thirty adults, aged 19–39, participated in the study, of which 10 participants were excluded for excessive missing values or contradictory responses (see below in the “Results” section). All participants were sexually active (had at least one intercourse over the past 6 months) and were not in a stable relationship. The participants were recruited through advertisements in the social media “Facebook,” by different experimenters and from two separated locations .All participants filled out an online informed consent form and were given the opportunity to participate in a lottery to win 500 NIS (∼125 euros) by leaving an email address. The following questionnaires were completed by the participants:The participants provided background information on age, gender, education, religiousness level, and sexual orientation.The Sexual Risk-Taking Behavior Scale (SRTB)  to 5 (very often). The questionnaire has high internal consistency (α = 0.88). The scale’s sensitivity and specificity are 68.4 and 99.6%, respectively (p = 0.6–0.9), high internal consistency of α = 0.89, and sensitivity and specificity of 62.7 and 68%, respectively   is a dimectively . The Hebectively .In order to characterize the sample in terms of sexual history, participants provided information on sexual debut, lifetime number of sexual partners, history of sexually transmitted diseases, and HIV tests. In addition, participants reported on any history of a diagnosis of ADHD and the use of medications to treat ADHD.Total scores were calculated for each participant on each questionnaire. Using Weber’s regression equation , for eacTo test the first hypothesis, a regression analysis of the relations between ADHD symptoms and frequency of SRTB was computed while controlling for the general sexual activity variable. To test the second hypothesis, direct and indirect effects of ADHD symptoms on the likelihood of SRTB were calculated using the multiple mediation approach and SPSS macro  provided by Data from participants, which met the following criteria, were not included in the analyses: (1) missing values exceeded 30% in one or more of the scales (six participants). (2) Responses contradicted each other . When one contradiction was observed, only the contradicting items were removed from the database. When more than one contradiction was observed, the entire data of the participant were removed (four participants).n = 33 and 87, respectively). Mean age of the sample was 25.77 (±4.33). The sample consisted of 72.5% females; 90.8% of the participants had high education; 89.2% of the sample identified as non-religious; and 92.5% identified as heterosexual. Regarding diagnosis of and treatment for ADHD, 19.2% reported that they had been formally diagnosed with ADHD, and 14.2% of the sample reported ever using medication for ADHD. Regarding history of risky sexual behavior, 70.8% reported having three or more partners so far, 45.0% have ever been screened for STD with 5.0% of the sample found positive, 41.7% of the participants (or their partners) have ever used “morning after” pills, and 5.0% had unintended pregnancy.Participants were recruited from two locations: Tel Aviv and Jerusalem districts . Skewness and kurtosis tests were used to confirm the normality of distribution. Skewness indices of all scales, except for the frequency scale, were in the −1 to 1 range . Kurtosis indices of all scales, except for the benefit attitude, risk attitude, and frequency scales, were in the −1 to 1 range . As three out of six scales were not normally distributed, further analyses were conducted using non-parametric tests.M = 3.14, SD = 0.70, and M = 2.53, SD = 0.46, respectively, t(26.74) = 3.98, P < 0.001, Hedges’ g = 1.19]. Next, we analyzed the responses to specific items in the likelihood, frequency, and risk perception scales, under the assumption that their associated level of risk differs in an obvious way. Items 9a, b and 9c, d in the likelihood and risk perception levels  refer to sex with a new partner and differ in whether the sexual history of the partner is known. As expected, the likelihood and frequency scores were lower, and the risk perception score was higher when the history of the partner was unknown (P < 0.001). Similarly, items 10a, b and 10c, d in the likelihood and risk perception levels  refer to regular but non-committed sex and differ in whether the sexual history of the partner is known. As expected, the likelihood and frequency scores were lower, and the risk perception score was higher when the history of the partner was unknown (P < 0.001).Several analyses were conducted to support the trustworthiness of the responses and the validity of the scales. First, we compared ASRS mean scores between the participants who reported having and not having an ADHD diagnosis. As expected, the ASRS scores of the participants with a history of an ADHD diagnosis were significantly higher .The correlations between the variables included in the hypotheses and the demographic variables were examined. As presented in f2 = 0.035). Male gender, general sexual activity, and ASRS score predicted SRTB above and beyond all other predictors . The standardized regression coefficient between ADHD symptoms and SRTB before considering mediators, and between ADHD symptoms and benefit perception, was statistically significant. The bootstrapped standardized indirect effect mediated by benefit perception was significant and of moderate effect size. The indirect effects of ADHD symptoms, mediated by risk perception, risk attitude, and benefit attitude, were not significant. ADHD symptoms did not predict SRTB after accounting for the indirect effect  the level of ADHD symptoms in the general population correlates with the frequency of engagement in SRTB even after controlling for general engagement in sexual behavior; (b) a link between ADHD and SRTB exists through the positive correlation of ADHD and benefit perception.Self-reported ADHD symptoms predicted the self-reported real-life frequency and the hypothetical likelihood of engagement in SRTB. These findings are in agreement with other studies documenting increased SRTB by people with ADHD , as wellThe link between ADHD and increased engagement in SRTB may be explained by increased overall engagement in sexual behavior. To test this hypothesis, the reported frequency of the general sexual activity was used as a covariate. ADHD symptoms still predicted SRTB even after covariating for the general sexual activity.Importantly, the link between ADHD and risk-taking behavior is not limited to SRTB. Numerous studies consistently reported associations between ADHD and higher engagement in other risk-taking behaviors, e.g., substance use, reckless driving, and gambling  of engaging in SRTB. Interventions may be devised considering the research, which would deal with external regulation and strategies that consider the individuals’ perceptions.This study has several limitations: The sample size was not big enough to further examine factors that affect the link between ADHD and SRTB. The convenience sampling resulted in an over-representation of women and of individuals with higher education and with a history of ADHD diagnosis. However, the degree of education did not correlate with risk measures. In addition, engagement in SRTB was assessed using self-report, which was not validated by a collateral report.This study was carried out in accordance with the recommendations of the Declaration of Helsinki with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the ethics committee of the Seymour Fox School of Education, at The Hebrew University of Jerusalem.TS and YP designed the study and analyzed the data. TS collected the data and wrote the manuscript. YP reviewed and revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Executive functioning deficits are common in children with neurodevelopmental disorders. However, prior research mainly focused on clinical populations employing cross-sectional designs, impeding conclusions on temporal neurodevelopmental pathways. Here, we examined the prospective association of executive functioning with subsequent autism spectrum disorder (ASD) traits and attention-deficit/hyperactivity disorder (ADHD) traits.This study included young children from the Generation R Study, a general population birth cohort. The Brief Rating Inventory of Executive Function-Preschool Version was used to assess parent-reported behavioral executive functioning when the children were 4 years old. ASD traits were assessed at age 6 (n = 3938) using the parent-reported Social Responsiveness Scale. The Teacher Report Form was used to assess ADHD traits at age 7 (n = 2749). Children with high scores were screened to determine possible clinical ASD or ADHD diagnoses. We were able to confirm an ASD diagnosis for n = 56 children by retrieving their medical records and established an ADHD diagnosis for n = 194 children using the Diagnostic Interview Schedule for Children-Young Child version (DISC-YC). Data were analyzed using hierarchical linear and logistic regressions.Impaired executive functioning was associated with more ASD and ADHD traits across informants . Deficits in all subdomains were associated with higher levels of ASD traits, whereas only impaired inhibition, working memory, and planning/organization were associated with more ADHD traits.The findings of the current study suggest a graded association of executive functioning difficulties along the continuum of ASD and ADHD and that problems in executive functioning may be a precursor of ASD and ADHD traits from an early age onwards. Executive functions are a set of cognitive abilities that are needed for regulating behavior, including inhibition, working memory, and planning. The ability to regulate behavior is important, as executive functioning has a substantial impact on short-term and long-term life outcomes such as physical and mental health, performance in school, and socioeconomic status , 2. ExecAutism spectrum disorder is characterized by deficits in social interaction and communication, and restricted behavior and interests, whereas the main symptoms in ADHD are inattention and hyperactivity/impulsivity . The preExecutive functioning deficits associated with both ASD and ADHD are found consistently throughout the literature , 16, 17.Only a minority of studies in this field has focused on young children with neurodevelopmental traits. Young children with ADHD or at high risk for ADHD appear to be impaired in executive functioning –35, whilFurthermore, most of the previous studies employed cross-sectional designs, impeding any conclusions on timing and temporality of associations. In addition, clinical studies often only include children in the clinical range, disregarding the other end of the spectrum. However, population studies include children from the general population, representing the full continuum and allowing for analysis along the entire dimension of executive functioning, ASD and ADHD. Potentially, deficits in executive functioning may be an expression of the latent vulnerability to ASD and ADHD . A betteThe aim of the current study was to investigate the association of executive functioning at age 4 years with ASD and ADHD traits at age 6/7 years. Specifically, we wanted to determine whether executive functioning could be an early indicator of later neurodevelopmental traits, independent of pre-existing traits. For this, we used a behavioral measure of executive functioning assessed in a general population cohort to explore impairment across the continuum of ASD and ADHD. Based on existing research, we expected impaired overall executive functioning to be prospectively associated with greater levels of ASD and ADHD traits. First, we expected that all executive functioning subdomains are associated with ASD traits. Second, we expect that specific executive function subdomains, including difficulties with inhibition, working memory, and planning, are associated with ADHD traits.This study was embedded in the Generation R Study , a largeIn total, we had 4450 children in our sample whose parents all completed the executive functioning questionnaire and who had information available on at least one of the following three assessments: ASD traits as reported by parents (n = 3938), ADHD traits rated by the teacher (n = 2749), or ADHD symptoms acquired in a clinical interview conducted with parents (n = 777). Among these 4450 children were 56 with a clinician confirmed ASD diagnosis and 194 with an ADHD diagnosis established based on a clinical interview  through 2 (sometimes) to 3 (often). Higher scores indicate more difficulties in executive functions. The BRIEF-P consists of 63 items covering five subscales: inhibition (16 items), shifting (10 items), emotional control (10 items), working memory (17 items), and planning/organization (10 items). All subscales and the total score were used in the analyses. Internal consistency of the overall score and the five dimensions was high: total score α = .95, inhibition α = .88, shifting α = .81, emotional control α = .83, working memory α = .89, planning/organization α = .78.At age 4 years (SD = 1 month), executive functioning was assessed with the validated Brief Rating Inventory of Executive Function-Preschool Version (BRIEF-P) –46. The not true) to 2 (very true or often true), where higher scores indicate more problems. Here, we used the total problem score and the DSM-oriented ADHD subscale. The CBCL 1.5–5 has shown to be a reliable and valid measure for child emotional and behavioral problems , p < .001, Table p < .001, Table More executive functioning difficulties at age 4 were associated with higher levels of ASD traits at age 6  and for shifting and ADHD diagnosis .To easily compare the results on ASD and ADHD, Fig. This study found that impaired executive functioning at the age of 4 years was prospectively associated with ASD and ADHD traits 2–3 years later, independent of multiple confounders and pre-existing psychopathology. Difficulties across executive functioning domains were associated with higher levels of ASD traits, whereas only impaired inhibition, working memory, and planning/organization were associated with more traits of ADHD. Importantly, our findings were consistent across informants: mother-reported ASD traits and clinical ASD diagnoses yielded similar results, as did teacher-reported ADHD traits and ADHD diagnoses based on mother reports. When excluding children with an ASD or ADHD diagnosis from the analysis, we were able to confirm that this association is not fully driven by a subgroup with clinically relevant levels of ASD and ADHD traits, but that, importantly, the associations were also observed in children with sub-clinical levels of these traits. Therefore, our findings provide evidence for a graded association of executive function impairments along the continuum of ASD and ADHD. Due to the nature of our data, we cannot draw any causal conclusions. However, our results implicate future studies to add to our findings, examining the causality of this relationship more in depth.In line with several previous studies , 20, 25,Our study also showed that deficits in overall executive functioning were associated with higher levels of ADHD traits and with a greater likelihood of being diagnosed with ADHD. In line with most previous research, specific domains of executive functioning, inhibition, working memory, and planning/organization, were related to ADHD traits and likewise to ADHD diagnoses , 18. HowThe results of the current study support the notion that executive functioning deficits overlap considerably among neurodevelopmental disorders. A general psychopathology factor has indeed been identified by multiple studies , 67, sugDespite this evidence for an overlap of executive functioning deficits with ASD and ADHD symptoms, unique variance needs to be considered as well. Reviews on the neurobiology of ASD and ADHD show several differences , 74, sucExecutive dysfunction could be part of the broader phenotype of neurodevelopmental traits, but our findings also suggest other possibilities. The longitudinal design of this study suggests some developmental difference in the trajectory of symptoms: rather than being parallel to ASD and ADHD traits, executive functioning may precede traits of these neurodevelopmental disorders. The associations remained even after adjusting for baseline behavioral problems. It could potentially be that deficits in executive functioning worsen the expression of children’s ASD or ADHD traits and, reversely, perhaps good executive functioning skills can serve as a buffer, tempering the severity of developmental disorders . HoweverThe current study had several strengths. First, we examined the prospective relationship between executive functioning and neurodevelopmental disorders in very young children in a large cohort, enabling us to control for multiple confounding variables, importantly baseline emotional and behavioral problems of the children. Second, we used multiple informants in this study; namely mothers, teachers, and medical records, yielding largely consistent results across these raters. Finally, both clinical diagnoses as well as sub-threshold traits of ASD and ADHD were considered, which addresses the research questions across the neurodevelopmental continuum.Despite these strengths, multiple limitations need to be mentioned as well. First, the non-response analysis indicated that socially disadvantaged children who are at higher risk of psychiatric problems were more likely to drop out. However, this selective loss to follow-up seems to affect only prevalence estimates, while longitudinal relationships estimated by association analyses remain relatively unchanged . Second,Our findings suggest that early executive functioning impairments may be a precursor of neurodevelopmental problems at a later age, for both children with clinical as well as with sub-clinical traits of ASD and ADHD. This supports the idea that children in the sub-clinical range should not be forgotten, but rather should be able to receive help when needed. Moreover, although it is not our aim to propose changes to the diagnostic framework, our results could point towards a possibility of identifying and monitoring children early who are at risk for developing clinical ASD or ADHD or having greater severity of ASD or ADHD. This allows for early intervention, which can potentially help prevent children from having persisting difficulties in executive function, developing more severe neurodevelopmental problems, and having negative outcomes later in life.Additional file 1: Table S1. Correlations Between Predictor and Outcome Variables. Table S2. The Association Between Executive Functioning and ASD Traits After Removing Clinical Cases (n = 3731). Table S3. The Association Between Executive Functioning and ADHD Traits After Removing Clinical Cases (n = 2612).


Multiblock copolymers constitute a basis for an emerging class of nanomaterials that combine various functional properties with durability and enhanced mechanical characteristics. Our mini-review addresses synthetic approaches to the design of multiblock copolymers from unsaturated monomers and polymers using olefin metathesis reactions and other ways of chemical modification across double C=C bonds. The main techniques, actively developed during the last decade and discussed here, are the coupling of end-functionalized blocks, sequential ring-opening metathesis polymerization, and cross metathesis between unsaturated polymers, or macromolecular cross metathesis. The last topic attracts special interest due to its relative simplicity and broad opportunities to tailor the structure and hence the properties of the copolymer products. Whenever possible, we analyze the structure–property relations for multiblock copolymers and point to their possible practical applications. Nowadays, olefin metathesis has become a well-established field of organic and polymer chemistry. The discovery of metallocarbene initiators that are capable of catalyzing metathesis polymerization in a living fashion turned it into a powerful tool of polymer design . Hundred4910In this mini-review we consider the approaches to multiblock copolymer syntheses via olefin metathesis reactions developed mainly over the past ten years. The following sections address the achievements and perspectives of three main techniques used for this purpose, namely, sequential ring-opening metathesis polymerization, coupling of end-functionalized blocks, and macromolecular cross metathesis.Ð = Mw/Mn = 1.0–1.19) and enables control over the block sequence and length in the copolymer chains (sequence-controlled multiblock copolymers). However, in practice this method is restricted to copolymers with a limited number of blocks, such as tetrablocks or pentablocks . A polymers  64]..n-octylfThe ADMET technique was used not only for the synthesis of polymer telechelics but also for their assembling into multiblock copolymers. A simple one-pot way for the preparation of random multiblock copolymers was proposed in reference . A mixtu2 with a norbornenyl-containing initiator (An approach utilizing macromonomers or macrocycles was used for the synthesis of multiblock copolymers with random or sequence-controlled structure . The ROM6nitiator  68]. Th. Th2 witVarious linear and star-shaped (triarm) ABA and ABCBA amphiphilic multiblock copolymers containing acetal-protected sugars (APS) were prepared by the coupling of an end-functionalized ROMP copolymer of norbornene (NB) and APS-substituted NB with poly(ethylene glycol) (PEG) . Ring-opThe MCO was obtained by ring-closing metathesis and contained easily cleavable ester linkages. It gave the possibility to сut the multiblock copolymer into pieces under alkaline conditions in order to obtain telechelic polyoctenylene with carboxyl end groups. The last reaction represents an example of the so-called sacrificial synthesis, another effective approach to telechelics .Hiff and Kilbinger generated cleavable ABAB pentablock and ABABABA heptablock metathesis copolymers via the sequential ROMP of seven-membered cyclic acetals  and N-substituted NB dicarboximide derivatives . The sub1 or a pyridine-containing end-terminator to yield pyridine-functionalized telechelic polymers 2. On this basis, supramolecular copolymers with alternating blocks were constructed using AgBF4 to remove Cl from the pincer complex and generate a cationic Pd ligand, which can coordinate with pyridyl ligands in a new pincer complex.Supramolecular multiblock copolymers with the possibility to introduce stimuli-responsive functionalities were obtained using a bimetallic ruthenium initiator . The iniA range of Zr(IV) and Hf(IV)-based bisamido complexes can catalyze both ROMP and addition (AP or vinyl) (co)polymerization of NB –75. The E-stereoregular multiblock copolymers and heterotelechelic polymers were successfully synthesized by the cross metathesis between different ROMP-derived poly(3-substituted cyclooctenes),  . Th. ThE-stectenes),  87]. Th. ThE-stectenes),  88–93].–93.E-steJ mol−1) ,96 is thJ mol−1) 91,93. ObJ mol−1) .MCM is an interchain cross reaction characterized by reshuffling of monomer units in the macromolecular backbones via break up and formation of new double bonds according to the olefin metathesis mechanism. In the beginning, an exchange of chain segments between the parent homopolymers results in the formation of diblock copolymers. Then random multiblock copolymers are formed , their aTg values are observed which get closer and finally merge into one, when long sequences of chemically identical units stemming from the parent homopolymers are exhausted.Therefore, the copolymer chain structure can be controlled by altering the reaction time, molar ratio of the starting polymers, catalyst type and concentration, as well as solvent type and initial polymer concentration –8993. It891H NMR spectroscopy was implemented to track the evolution of the chain structure in the course of MCM between polybutadiene (PBD) or polyisoprene (PI) and olefin-containing polyesters or polyurethane, as well as changes in the chain stereospecificity during the reaction between 3-substituted PCOEs [13C NMR [91L was calculated from an integral ratio of homo  and heterodyad (A–B) signals in the NMR spectra:ed PCOEs –86. Cros[13C NMR –9193. ThLA = [I(CA–A)+I(CA–B)]/I(CA–B); LB = [I(CB–B)+I(CB–A)]/I(CB–A); where I(CA–A) and I(CB–B) are the peak intensities of the initial homodyads, A–A and B–B, and I(CA–B) and I(CB–A) are the peak intensities related to the alternating dyads.The average block lengths decreased with the conversion, reaction time, and catalyst concentration and asymptotically approached the value of 2, characteristic of a completely random (Bernoullian) equimolar copolymer. Thus, a proper choice of the MCM conditions enables one to obtain copolymers with a controllable average block length ranging from the initial homopolymer length to a few monomer units.Important data on the kinetics of MCM between PNB and PCOE mediated by Gr1 were obtained by combining in situ NMR studies of the Ru-carbene transformations and ex situ NMR monitoring of the dyad composition evolution . It was It takes about a day for the interchain exchange between the homopolymers with carbene-functionalized end groups to yield a statistical NB–COE copolymer and during this process its molar mass remains almost unchanged. The slowest elementary reaction, which controls the overall kinetics, is the interaction between [Ru]=PCOE carbenes and C=C bonds in PNB chains (An increase of the PNB concentration in the mixture results in a growth of the copolymer degree of blockiness . This fecis/trans-isomerization of double bonds in the MCM process were obtained . It. It3Si-ss formed –88. Besis formed  via douboic acid . It was trans-octenylene blocks. Compared with the pure PCOE or its equimolar blend with PNB, the NB–COE copolymers form considerably smaller crystallites (Copolymer crystallinity can be studied in detail by combining WAXD and DSC methods, including recently emerged technique of thermal fractionation by successive self-nucleation and annealing ,102–103.tallites  92]..trans-ocIt is rather clear nowadays that the olefin-metathesis reaction is a versatile tool for the synthesis of multiblock copolymers with diverse chemical structures. Due to the rapid progress in the catalyst design for living polymerization, sequential ROMP has become a well-established method of obtaining copolymers with sequence-defined structures. However, in many aspects, this technique remains laborious and even cumbersome. Most publications report on the multiblock copolymers synthesis by the coupling of premade individual blocks. A key point here is related to advances in the development of synthetic approaches for fabricating symmetric and asymmetric telechelics and monochelics, macromonomers and macrocycles based on different olefin-metathesis techniques like CTA, ADMET, etc. A subsequent assembling of macroblocks into copolymers can be carried out by combining olefin metathesis with other reactions such as ATRP, RAFT, click-reaction, and so on, which permit to gain certain control over the final copolymer structures. The most recent approach to the multiblock copolymer synthesis implements the macromolecular cross-metathesis reaction, which is still poorly studied. For this method, the simplicity of realization is counterweighted by inability of precise control over block sequences and considerable drop in the average molecular mass of reacting polymers as a result of their interaction with metathesis catalysts. Nevertheless, the average block lengths can be easily tailored and the resulting copolymers reveal the ability to self-assemble into ordered structures, enhanced mechanical properties, and nontrivial crystalline and thermal characteristics. Recent kinetic studies with the use of in situ and ex situ NMR have shed some light on the regularities of the macromolecular cross-metathesis reaction, which appeared to be somewhat opposite to the notions about metathesis copolymerization. Perspectives of the entire field under review are related to the elaboration of novel post-modification methods for obtaining new functionalities and enhancing various characteristics of multiblock copolymers. In our opinion, further development of the olefin metathesis methods for the multiblock copolymer synthesis will be directed by the search for new properties and possible applications.


Multiple logistic regression analyses were used to find those parental risk factors of LBW by comparing two groups. TLBW and TNBW(50th–97th) occupied 4.8% and 70.8% of the study population, respectively. Logistic regression showed a significant association with positive maternal hepatitis B surface antigen , irregular folic acid intake , paternal history of varicocele  and female babies . Maternal smoking, hypertension and history of stillbirth were found related to LBW but no statistically significant. Positive maternal hepatitis B surface antigen, irregular folic acid intake, paternal history of varicocele had a negative effect on birth weight. Measures are necessarily taken to avoid them to improve pregnancy outcomes. Further studies should be done to investigate each detailed risk factors on LBW.No large population-based study has focused on both maternal paternal risk factors for low birthweight (LBW) in China. We aimed to identify parental risk factors associated with LBW.A population-based, retrospective cohort study was conducted on 202,725 singleton infants at 37–42 weeks. These term singleton newborns were classified as LBW with birthweight ≤2500 g(TLBW) and normal birthweight between 50 The definition of low birthweight(LBW) infant was recommended as infant ≤2500 g regardless of gestational age by The World Health Organization(WHO) in 19502. The incidence of LBW was 6.1% in mainland China reported by Chen Y et al.3 and 8.1% in Foshan of China by Rao J et al.4. The risk factors of LBW become important in lowering the incidence of LBW. With the support of National Health and Family Planning Commission of China(NPCP), our study covered 60% pilot counties with over 200,000 population in total. Although several studies concluded some common maternal risk factors associated with LBW, few studies included paternal factors. Some experts started to suggest the consideration of paternal information in studies aimed at the risk factors model of LBW. We are the first large study to analyze both maternal and paternal risk factors of LBW in China.Birth weight is a convenient factor indicating adverse outcomes of infants. Low birthweight infant has a higher perinatal mortality and it also increases health risk in adulthoodth–97th (TNBW 50th–97th) accounted for 70.8% and it had the highest mean birthweight (3454.34 ± 297.76 g). The remaining term babies were 31432 with the mean birthweight was 2855 ± 147.66 g. Figure 202,725 cases were analyzed in our study, the prevalence of term low birthweight infants(TLBW) was 4.8% (n = 9687) in rural China with the mean birthweight was 2140.54 ± 402.53 g. Among term low birthweight babies, 647 of them were very low birthweight infants. Term infants between 50th–97th) group, maternal hypertension and smoking in our study had a negative effect on birthweight of infants. However, it was no statistically significant. Maternal positive hepatitis B surface antigen was associated with decreased birthweight in our study . Birthweight in regular folic acid group was higher than irregular group . Additionally, Paternal history of varicocele would significantly increase the risk of LBW . Female babies had a little higher risk of low birthweight  , red blood cell count, positive hepatitis B surface antigen, gestational age and paternal weight, height, BMI, age, history of varicocele and female infant were found significant in univariate analysis. Table 5. But it is still not rare in developing countries with the overall prevalence was 15.9%6. To lower the incidence of LBW, it is very important to identify risk factors. Research have found maternal age, education level, BMI, ethnicity, socio-economic level, medical disease before and during pregnancy, health care and traffic air and noise pollution were related to LBW8. We found several important maternal and paternal risk factors which may involve in the process of LBW by a large, population-based database. Several studies have reported maternal hypertension and smoking were associated with LBW. Our current study showed the prevalence of LBW in maternal hypertension and smoking group were higher in those without hypertension and smoking before pregnancy but with no statistical significance in multiple risk analysis. Nevertheless, the number of rural smoked mothers were too small and many of them exposed to second-hand smoke which may mix the results. Interestingly, maternal exposure of hepatitis B surface antigen was found to be related to TLBW. Recent research also showed positive HBsAg may lead to a high risk of malformation and cesarean delivery but lower risk of macrosomia and non-significant higher risk of LBW in China9. The mechanism may be related to reduced blood supply of uteroplacenta due to the infection. This conclusion addressed the importance of management of maternal HBsAg carrier status. Several studies had demonstrated that folic acid supplementation was associated with decreased lowbirthweight infants. We found LBW easily happened in irregular folic acid intake group10. Maternal anemia is known to be related to folic acid supplementation, which will cause fetal hypoxia and intrauterine fetal distress11. Maternal anemia during pregnancy is an independent risk factor for low birthweight and preterm delivery. We are the first study found the relationship between low birthweight and paternal history of varicocele. Christman MS et al. tested the lower seman density and count in patients with a history of varicocele and cryptorchidism. We guess it is the main reason lead to LBW by zygotes with poor quality12. Further studies need to be done to address the mechanism. Our study also had limitations. Some of our data source were derived from questionnaire which lacked quantitative indices. Further, this study was conducted in rural area of China, so it was difficult to obtain the data covered whole China.LBW is associated with increased risk of diabetes mellitus, hypertension and cognitive dysfunction later during lifeOur study demonstrated that both maternal and paternal factors could affect birth weight. Maternal smoking and hypertension would lead to a non-significant LBW. Maternal positive hepatitis B surface antigen and irregular folic acid intake and paternal history of varicocele would increase the risk of LBW. In conclusion, more effort should be made to improve pre-pregnancy checkups to prevent LBW and long-term outcomes.15. Our study included 248,501 records, among which ectopic pregnancy occurred in 176 cases and spontaneous abortion and self-induced abortion occurred in 5714 and 2526 pregnant women, respectively. 786 of records were stillbirths. 230,190 live births at 37–42 weeks were included, twins, triplets and those infants with missed birth information were first excluded. Full-term infants whose birthweight ≥97th percentile according to Chinese birthweight percentile norm for newborn were then excluded16. Finally, 202,725 single live births at 37–42 weeks were included in our study  organized by The Population and Family Planning Commission and The Ministry of Finance of China between January, 2010 and December, 2013. It covered rural areas of 30 provinces of China: Beijing, Hebei, Tianjin, Shandong, Zhejiang, Jiangsu, Anhui, Jilin, Fujian, Jiangxi, Henan, Hubei, Guangdong, Sichuan, Chongqing, Yunnan, Shanxi, Shanghai, Guangxi, Hunan, Hainan, Heilongjiang, Qinghai, Tibet, Inner Mongolia, Shanxi, Liaoning, Ningxia, Guizhou and Gansu. This project was built for couples planning on getting pregnant in rural China. Each couple will receive general information and medical history inquiry, physical examination, clinical laboratory examination and imaging examination. Included couples will be followed up on 12th weeks of gestation and 6 weeks after delivery. Last menstrual period was used to calculate the gestational age. Infants at 37–42 weeks were defined as term newborn. The group of NPCP has already worked on the risk factos of adverse pregnancy outcomesThe medical staffs from NPCP were trained by professional experts before collecting data and the database was built by application developers. To ensure the accuracy of data, questionnaires were completed by two trained interviewers at the same time. Our database had items including social demographics, illness and medical history, living habits, psychological status, environmental exposure, maternal pregnancy complications and biological parameters of parents Table .Table 2Ith–97th (TNBW 50th–97th): Infants between 50th–97th percentile with boy’s birthweight between 3073 g to 4410 g and girl’s birthweight between 2964 g to 4212 g. (3) The remaining infants. We included the following variable in our analysis including categorical variables and continuous variables. Table All included infants were divided into three groups: (1). Term low birthweight (TLBW): birthweight ≤2500 g; (2). Term normal birthweight from 50The informed consent was obtained from all subjects and all methods were carried out in accordance with relevant guidelines and regulations. All experimental protocols were approved by NPCP.17. P < 0.05 was considered statistically significant. SPSS 24.0 edition was used for data analysis.Continuous data were performed as the mean and standard deviation (SD) and categorical data were performed as frequency and percentage. The chi-squared test was used for qualitative data and independent sample t-test were used for quantitative data analysis. Variables with P value <0.1 was considered as significant in screening single factors. To address confounding variables, stratified analysis on all suspected confounders was used to estimate the specific risk for each stratum, stepwise multiple logistic regression was performed to correct the confounders in multi-factor analysis if the ajusted effect differs between each subgroup


Melanocortin 3 receptor (MC3R), a rhodopsin-like G protein-coupled receptor, is an important regulator of metabolism. Although MC3R knock-out (KO) mice and rats were generated in earlier studies, the function of MC3R remains elusive. Since pig models have many advantages over rodents in metabolism research, we generated an MC3R-KO pig using a CRSPR/Cas9-based system combined with somatic cell nuclear transfer (SCNT) technology.Four CRSPR/Cas9 target vectors were constructed and then their cleavage efficiency was tested in porcine fetal fibroblasts (PFFs). The pX330-sgRNA1 and pX330-sgRNA4 vectors were used to co-transfect PFFs to obtain positive colonies. PCR screening and sequencing were conducted to identify the genotype of the colonies. The biallelically modified colonies and wild-type control colonies were used simultaneously as donor cells for SCNT. A total of 1203 reconstructed embryos were transferred into 6 surrogates, of which one became pregnant. The genotypes of the resulting piglets were determined by PCR and sequencing, and off-target effects in the MC3R KO piglets were detected by sequencing. Then, offspring were obtained through breeding and six male KO pigs were used for the growth performance analysis.−/− and 10 MC3R+/− clones, were obtained, corresponding to a gene targeting efficiency of 29.17%, with 15.28% biallelic mutations. A total of 6 piglets were born, and only two MC3R KO piglets were generated, one with malformations and a healthy one. No off-target effects were detected by sequencing in the healthy mutant. Six male MC3R KO pigs were obtained in the F2 generation and their body weight and body fat were both increased compared to wild-type full siblings.Four vectors were constructed successfully, and their cleavage efficiencies were 27.96, 44.89, 32.72 and 38.86%, respectively. A total of 21 mutant colonies, including 11 MC3RA MC3R KO pig strain was generated using the CRSIPR/Cas9-based system, which makes it possible to study the biological function of MC3R in a non-rodent model.The online version of this article (10.1186/s12944-019-1073-9) contains supplementary material, which is available to authorized users. The melanocortin receptor (MCR) family belongs to the rhodopsin-like G protein-coupled receptor family with seven conserved domains, and plays a pivotal physiological role in energy homeostasis . MelanocCurrently, pigs are extensively used as a large-animal model for biomedical research because of their greater similarities with humans in terms of anatomy, physiology, and immunology, compared to rodents , 16, 17.In this study, MC3R KO piglets were successfully generated using the CRISPR/Cas9 system combined with somatic cell nuclear transfer (SCNT) technology, providing a valuable non-rodent model to elucidate the physiological function of MC3R in the future.The experimental procedures used in this study, including the feeding, transport, sampling and surgery of the subject pigs, were approved by the Jiaxing University Experimental Animal Welfare Ethics Committee. All surgical procedures were performed under anesthesia, and all efforts were made to minimize animal suffering.BbsI sites), pEGFP-N1 (including eGFP) and pL425 (containing a neomycin resistance gene) were obtained from Addgene. Four sgRNAs  were selected in MC3R using CRISPR/Cas9 Design software (http://crispr.mit.edu/), targeting the CDS, 3′ UTR and 5′ UTR of MC3R in the pig genome  were isolated from 30-day-old fetuses of Chinese Experimental Mini (CEM) pigs. The heads, tails, limbs and viscera of the fetuses were removed aseptically, and the remaining tissue was cultured in high glucose DMEM  with 15% FBS  and 1 × Penn/Strep solution  using previously described methods. Isolated PFFs were cultured in 10-cm dishes for 12 h and frozen in cell freezing medium . A day before transfection, PFFs were thawed and cultured to sub-confluency. Aliquots comprising 1 μg of the To identify the large-fragment deletion in the pig genome, MC3R targeting vectors (pX330-sgRNA1 and pX330-sgRNA4) and pL452 (expressing neomycin resistance) were introduced into PFFs, and the cells were seeded into 10-cm dishes. After 48 h, 800 μg/mL of G418  was added to select single colonies for 8–12 days. One half of the single colonies was cultured in 24-well plates for PCR screening, and the other half was transferred into 12-well plates for SCNT. The PCR screening primers used to amplify the target region were as follows: Forward: 5′-ATCTCTCAAGGGGTGTCTCCCG-3′, reverse: 5′- TGTTCTCAGGTGACCGCA TGAC-3′. The PCR conditions were as follows: 94 °C for 2 min, followed by 30 cycles of 95 °C for 10 s, 60 °C for 30 s, and 72 °C for 5 min, and a final 72 °C for 10 min. The PCR products were sequenced  after ligation into a pMD19-T vector  according to the manufacturer’s instructions. Ninety-six individual clones were picked and sequenced.The oocytes for SCNT were collected from ovaries purchased from a local slaughterhouse, cultured for 42–44 h in vitro, and enucleated as described elsewhere . BrieflyThe genomic DNA of the F0 piglets was isolated from an ear biopsy, and the genomic region surrounding the target site was amplified by PCR. The PCR conditions were as follows: 95 °C for 5 min, followed by 30 cycles of 95 °C for 10 s, 60 °C for 30 s, and 72 °C for 40 s, and a final 72 °C for 7 min. The PCR products were sequenced  after insertion into a pMD19-T vector.Potential off-target sites (OTSs) were predicted using CRISPR Design software, amplified form the genomic DNA of the MC3R KO piglet, and sequenced. Variants were identified by sequence alignment. Sequences of the potential OTSs are shown in Fig. +/-) were mated with five sows (MC3R+/-) to produce the F2 generation. Six males with MC3R−/− genotype were selected for body weight and adiposity analysis, and compared with their full siblings with MC3R+/+ genotype.The MC3R KO piglet was mated with wild-type sows at eight months old. Eighteen piglets were obtained from three mothers at the F1 generation. Then, five male pigs  of Chinese Experimental Mini (CEM) pigs were used as donor cells for the generation of MC3R-knockout pigs. Since single nucleotide polymorphisms (SNPs) can potentially affect CRISPR/Cas9 efficiency, the genomic sequence of the MC3R locus in the CEM pig was analyzed by sequencing. Seven SNPs were found to differ from the MC3R sequence from GenBank  cell line with a large-fragment deletion. The pX330-sgRNA1 and pX330-sgRNA4 targeting vectors were used together with the pL452 vector (expressing neomycin resistance) to co-transfect early-passage primary PFFs. A total of 72 colonies were isolated after G418 selection for 10 days. Genotype analysis of each colony was performed using TA-cloning and sequencing. The results indicated that 21 out of the 72 colonies harbored a homozygous/heterozygous biallelic mutation in the MC3R target gene region, including 11 MC3R−/− knockouts . A total of 1203 reconstructed embryos were transferred into 6 surrogates. B-ultrasonic examination was performed on each recipient about 30 days after surgery, and 1 of the 6 gilts was found to be pregnant, corresponding to a pregnancy rate of 16.7% .