Purpose: The presence of a disorder of sexual development (DSD) acts as a diagnostic specifier for gender dysphoria (GD) under DSM-5, while the International Classification of Diseases (ICD)-10 specifically states that its equivalent diagnosis, gender identity disorder (GID), must not be the result of a chromosomal abnormality. For these reasons, routine karyotyping has been previously advocated in the clinical work-up of children and adolescents with suspected GD or GID. However, the utility of such testing remains unclear.Methods: The results of routine molecular karyotyping were analyzed in 128 patients attending our Australian statewide pediatric gender service from 2013 to 2016. Karyotyping was performed using an Illumina BeadChip platform and provided information on both sex chromosome composition and copy number variation (CNV).Results: No sex chromosome abnormalities directly suggestive of a DSD were discovered. The rate of CNVs among our patient cohort was 8.6% (11/128), similar to that previously reported for the general population. Unexpectedly, three trans male patients shared the same CNV, involving an almost identical 400 kbp deletion on chromosome 15q11.2. The frequency of this deletion within birth-assigned females in our cohort  was significantly higher than that within local control populations , suggesting a possible association between 15q11.2 deletions and trans male identity.Conclusion: Routine molecular karyotyping failed to detect any occult DSD and indicated that the rate of CNVs was similar to that of the general population. Given these findings, we suggest that molecular karyotyping has minimal clinical utility in the routine management of children and adolescents with GD. In such cases, the diagnosis of GD may therefore precede suspicion of a DSD, especially in individuals whose gender identity concerns manifest early in life.Thus, when assessing an individual with suspected GD for the first time, it is important to know whether he/she also has a DSD. In the vast majority of cases where GD and a DSD coexist, this is relatively straightforward since the diagnosis of a DSD has already been made.How then can such individuals with an occult DSD be identified when assessing for possible GD? In BAFs, a history of amenorrhea may be suggestive of either CAIS or XY gonadal dysgenesis , while physical examination may reveal reduced/absent pubic or axillary hair in both these conditions. In postpubertal BAMs, a history of infertility, gynecomastia, delayed puberty, as well as speech and learning difficulties may be indicative of KS, while physical examination may reveal increased height, reduced body hair and small testicular size in such individuals. However, in prepubertal children, history and examination are likely to be unrewarding, unless there is a history of inguinal hernia or speech and learning difficulties. One potential means to identify individuals with an occult DSD is karyotyping, which evaluates chromosomal composition. For example, in BAFs with occult CAIS or XY gonadal dysgenesis, karyotyping will signal the presence of an underlying DSD by revealing XY instead of XX chromosomes, while in BAMs with occult KS, karyotyping will reveal XXY instead of XY chromosomes.9 molecular karyotyping allows for the identification of genetic anomalies known as copy number variations (CNVs), which can include small chromosomal deletions, duplications, and rearrangements. Although there is no empirical evidence to suggest that there is an increased incidence of CNVs in individuals with GD, it is now well recognized that GD commonly coexists with Autism Spectrum Disorder (ASD),10–14 a neurodevelopmental condition that results in impaired social communication and behavior1 and is known to be associated with an increased risk of pathogenic CNVs.15 Given the latter, the American Academy of Pediatrics now recommends that molecular karyotyping be offered to all patients with ASD.16 Whether such testing might also be clinically useful for children and young people with GD—∼50% of whom score in the clinical range for ASD, using the Social Responsiveness Scale screening tool13—is unclear.Performing karyotyping in the context of a medical assessment for GD may be of potential value for other reasons. Molecular karyotyping, which uses genetic technology known as a chromosomal microarray, provides detailed genomic information at a much higher resolution than cytogenetic “G banding” techniques used in conventional karyotyping. Having superseded conventional karyotyping in clinical care in recent years,17 Thus, it could be argued that karyotyping should be routinely performed in the diagnostic workup of individuals with GD (or GID), and indeed this is commonly what is done in many countries even though it is not explicitly part of current international clinical guidelines.19In addition to the DSM, the World Health Organization's International Classification of Diseases (ICD) is the other standard tool for the diagnosis of GD. In the most recent version (ICD-10), gender identity disorder (GID)—as GD is referred to—has three criteria, one of which specifically states that GID must not be the symptom of a chromosomal abnormality.20–23 Taken together, these studies indicate an overall rate of chromosomal abnormalities in 4/286 trans males (1.4%) and 11/481 trans females (2.3%), both of which are higher than the reported 0.5% prevalence of abnormalities within the general population using conventional karyotyping techniques.24 Of the observed abnormalities, six involved sex chromosome abnormalities. Of these, four individuals had KS , one had mosaic Turner syndrome , and the remaining was mosaic 47XYY/46XY. Given the low prevalence of chromosomal abnormalities in transgender individuals observed in these studies, the prevailing recommendation was that the routine use of conventional karyotyping was of limited clinical utility and that karyotyping should be reserved for cases where suspicions of a DSD are raised based on other clinical features.Despite this, empirical data on the utility of karyotyping in the management of GD are limited. Between 2003 and 2013, five separate studies of transgender adults using conventional G banding techniques were reported .18,20–2323 Nevertheless, the clinical utility of karyotype testing in a pediatric population with suspected GD is currently unknown. Moreover, since previously reported studies in adult transgender populations have all used conventional G banding techniques to assess karyotype, it is also unclear whether chromosomal microarrays—which provide ≥10-fold improvement in resolution9—might provide additional clinically useful information, especially in light of the increased rates of ASD among those with GD.However, such recommendations do not necessarily apply to a pediatric population, in whom—for reasons already noted above—clinical evidence of some DSDs may not yet be evident. Indeed, on this basis, it has been advocated that the routine use of karyotyping to screen for DSDs should be performed in pediatric cases of suspected GD.The Royal Children's Hospital Gender Service (RCHGS) services a population of over 5 million people in the state of Victoria, Australia. From 2013, young people presenting to the service with suspected GD have routinely been offered molecular karyotyping to analyze chromosomal composition and evaluate for the presence of an occult DSD. Given the current gaps in knowledge, the aim of this study was therefore to determine the clinical utility of molecular karyotyping in a pediatric population with suspected GD.All children and adolescents presenting to the RCHGS from 2013 to 2016 with suspected GD were assessed by a mental health practitioner (either a psychologist or psychiatrist). In those cases where puberty was likely to be imminent or had already commenced, individuals were also seen by a pediatrician within the service. As part of the pediatric assessment, patients routinely received a physical examination (including Tanner staging) as well as hormonal measurement  and were offered molecular karyotyping to analyze chromosomal composition and evaluate for the presence of an occult DSD.Molecular karyotyping was performed on blood samples at the Victorian Clinical Genetics Service (VCGS) using Illumina CytoSNP or CoreExome BeadChip platforms, which interrogate between 300,000 and 500,000 single-nucleotide polymorphisms across the genome. Molecular karyotyping is the default method of karyotyping at VCGS and was provided at no cost to the patients.p-values to determine whether the 15q11.2 deletion was overrepresented within our patient cohort compared to published and local control groups, respectively. To determine whether the average ages of BAFs and BAMs were significantly different, a two-tailed p-value was calculated using Student's t-test. In each case, an α of 0.05 was used as a cutoff for significance. Ethics approval for conducting this audit was granted by the Royal Children's Hospital Human Research Ethics Committee.Molecular karyotyping results were assessed via retrospective audit by cross-referencing known patients of the RCHGS between 2013 and 2016 with VCGS molecular karyotyping records. To compare the prevalence of 15q11.2 deletions within our patient population and that of a local control group, VCGS molecular karyotyping records were also extracted from 4869 abortus material samples that were obtained from either fetal tissue or placental tissue of fetal origin and referred to VCGS for molecular karyotyping due to spontaneous miscarriage or abnormal fetal morphology. Chi-square with Yates correction and Fisher's exact test were used to calculate two-tailed t-test, p<0.05) at the time of testing. Out of 128, 112 individuals were subsequently diagnosed with GD based on DSM-5 criteria by their treating clinicians. For those 16 cases not diagnosed with GD, some failed to attend sufficient appointments to have the diagnosis confirmed, some were continuing to explore and/or were unsure of their gender identity, while others did not have GD.One hundred and twenty-eight molecular karyotypes were extracted from 69 BAFs and 59 BAMs. BAFs were significantly older (mean age: 14.7±3.3 years) than the BAMs  of our patients, the molecular karyotype was normal. This meant that the rate of CNVs observed within our patient cohort was 8.6% (11/128). Of the 11 abnormal karyotypes detected , 5 were 25 Consistent with this, our particular patient had previously been diagnosed with global developmental delay, ADHD, and ODD, was suspected of having ASD, and was subsequently noted to have facial features in keeping with those described in others with a 22q11.21 duplication.Only one of these was classified as a pathogenic change, and involved a 2.6 Mbp duplication on chromosome 22 (22q11.21). Similar duplications have been associated with a variety of clinical features, including global developmental delay, ASD, attention-deficit hyperactivity disorder (ADHD), oppositional defiant disorder (ODD), and dysmorphic facial features, with variable expressivity.26 Notably, the rate observed within not only our BAFs  but also our entire patient cohort  was significantly higher than within these controls . However, since the incidence of 15q11.2 deletions can vary between different populations, we also compared our observed rates with that of a local control group. This group consisted of abortus material samples referred to VCGS for molecular karyotyping due to spontaneous miscarriage or abnormal fetal morphology, and 16/4869 (0.3%) had 15q11.2 deletions, which was again significantly different to the rate observed among our patients . Taken together, these data raise the possibility that 15q11.2 deletions might confer susceptibility to GD and, more specifically, trans male identity.Four other CNVs observed within our patients were reported as being of “uncertain significance.” In each of these cases, the CNV had been previously associated with a range of clinical phenotypes that displayed incomplete penetrance . One of these CNVs was located on chromosome 22q11.21—similar to that of our other patient—but this one involved a deletion rather than a duplication. Unexpectedly, three of the other CNVs of “uncertain significance”—all of which were present in BAFs with a male gender identity—involved almost identical 400 kbp deletions on chromosome 15q11.2. In previous studies, this deletion has been found to occur at an overall rate of 0.3% in healthy control populations .The remaining four CNVs were not previously associated with any clinical phenotype and were therefore reported as being of “unknown significance.”27–32 With population-based surveys now estimating that 1.2% of teenagers identify as transgender,33 it is quite likely that referral rates will continue to grow. Knowing how best to assess and treat children and adolescents with suspected GD is therefore of increasing relevance to pediatric practice. In this regard, previous authors have suggested that karyotyping to screen for DSDs should be routinely performed in pediatric cases of suspected GD,23 but the clinical utility of doing so remains unknown.The number of children and adolescents being referred for specialist medical care with suspected GD has increased dramatically in recent years.34 our patient was genetically 46XY and showed no clinical evidence of a DSD, suggesting that the duplication is simply an incidental finding. Taken together, our results are therefore consistent with earlier studies of transgender adults,20–23 which suggested that routine karyotyping was of limited clinical benefit in identifying occult DSDs.In this study, we present for the first time molecular karyotyping results for a large cohort of children and adolescents presenting with suspected GD. Our routine molecular karyotyping of 128 children and adolescents failed to detect any occult DSD. Although one BAM displayed structural variation at a chromosomal locus (Xq26.3) that has been previously implicated in XX sex reversal,9 Although the precision of such methods carries potential disadvantages , our use of molecular karyotyping was potentially important for two main reasons, both of which related to a desire to explore the genetic basis of GD, support for which comes from previous twin studies.35–37Unlike previous karyotyping studies in transgender adults, our study used molecular karyotyping methods that provide much higher resolution than conventional G-banding techniques and can thus detect small chromosomal changes that are otherwise missed with conventional karyotyping.10 suggesting that there might be a shared developmental vulnerability underlying the two conditions. With this in mind—and given that ASD is known to be associated with an increased risk of pathogenic CNVs15—we hypothesized that molecular karyotyping might similarly reveal an increased rate of pathogenic CNVs in our patients. However, only 1/128 karyotypes (0.8%) was reported as containing a pathogenic CNV, which is equivalent to the rate expected within the general population.38 And, even though the discovery of this particular CNV was clinically useful , the individual involved had multiple clinical features that were suggestive of an underlying genetic cause  and should have been an independent prompt for molecular karyotyping. Thus, we would argue that our results do not support a role for routine molecular karyotyping as a screen for pathogenic CNVs in suspected GD, but that molecular karyotyping be offered in a targeted manner to patients with comorbid and unexplained intellectual disability, global developmental delay, and/or ASD, consistent with existing clinical recommendations for a general pediatric population.16First, as noted earlier, individuals with GD show much higher rates of ASD than expected by chance,38 we unexpectedly found almost identical deletions at chromosome 15q11.2 in three trans male patients. Deletion of this region disrupts four genes, each of which are highly expressed in the brain, and confers susceptibility to a range of neurodevelopmental disorders, including developmental delay, ASD, schizophrenia, and epilepsy.39 Given that the frequency of 15q11.2 deletions within our patient cohort was significantly higher than that of controls, our findings provide for the first time evidence that this deletion might increase susceptibility to a trans male identity and, more generally, highlight the potential advantages of adopting an unbiased genome-wide approach to understanding gender identity. After all, for over a decade, multiple groups have attempted to explore the genetic basis of transgender identity by looking at variants within specific candidate genes , but no consistent associations have been found.40–45Second, high-resolution molecular karyotyping offered an opportunity to explore whether any particular CNVs—regardless of whether or not they were reported as pathogenic—might be associated with GD. Interestingly, even though the overall rate of CNVs within our patient cohort (8.6%) was no higher than that previously observed within the general population,Finally, it is important to acknowledge the limitations of our study, which principally center on the relatively low number of molecular karyotypes that were assessed. Specifically, having only analyzed 128 molecular karyotypes, we cannot exclude the possibility that occult DSDs might be usefully detected at very low frequency with this technology. Nevertheless, many such DSDs will result in an abnormal hormonal profile during puberty, so endocrine evaluation is likely to provide an alternative and less expensive screening option in this regard. Our relatively low numbers similarly limited the power of our study to detect specific CNVs associated with GD and, in this regard, our finding of a significant association between a trans male identity and 15q11.2 deletion was quite unexpected. However, given our association signal was derived from just 3/69 BAFs, it will be critical to see whether this apparent association can be confirmed in future research studies using larger patient cohorts.Routine molecular karyotyping of 128 children and adolescents presenting with suspected GD failed to detect any occult DSD and indicated that the rate of CNVs was similar to that of the general population. We therefore suggest that molecular karyotyping has minimal clinical utility in the routine management of GD.


Background: Around 30% Parkinson's disease (PD) patients develop impulse control disorders (ICDs) to D2/3 dopamine agonists and, to a lesser extent, levodopa. We aim to investigate striatal dopaminergic function in PD patients with and without ICD.Methods: PubMed, Science Direct, EBSCO, and ISI Web of Science databases were searched  to identify PET or SPECT studies reporting striatal dopaminergic function in PD patients with ICD (ICD+) compared to those without ICD (ICD–). Studies which included drug naïve patients, explored non-pharmacological procedures , and those using brain blood perfusion or non-dopaminergic markers were excluded. Standardized mean difference (SDM) was used and random-effect models were applied. Separate meta-analyses were performed for dopamine transporter level, dopamine release, and dopamine receptors availability in the putamen, caudate, dorsal, and ventral striatum.Results: A total of 238 studies were title and abstract screened, of which 19 full-texts were assessed. Nine studies  were included in the analysis. ICD+ showed a significant reduction of dopamine transporter binding in the putamen , caudate  and dorsal striatum , and increased dopamine release to reward-related stimuli/gambling tasks in the ventral striatum . Dopamine receptors availability did not differ between groups. Heterogeneity was low for dopamine transporter in the dorsal striatum (I2 = 0%), putamen (I2 = 0%) and caudate (I2 = 0%), and pre-synaptic dopamine release in the dorsal (I2 = 0%) and ventral striatum (I2 = 0%); heterogeneity was high for dopamine transporter levels in the ventral striatum (I2 = 80%), and for dopamine receptors availability in the ventral (I2 = 89%) and dorsal (I2 = 86%) striatum, putamen (I2 = 93%), and caudate (I2 = 71%).Conclusions: ICD+ patients show lower dopaminergic transporter levels in the dorsal striatum and increased dopamine release in the ventral striatum when engaged in reward-related stimuli/gambling tasks. This dopaminergic imbalance might represent a biological substrate for ICD in PD. Adequately powered longitudinal studies with drug naïve patients are needed to understand whether these changes may represent biomarkers of premorbid vulnerability to ICD. Impulse control disorders (ICD), such as pathological gambling, hypersexuality, binge-eating, and compulsive shopping are diagnosed in around 30% of patients with Parkinson's disease (PD) –4.2/3 dopamine agonist treatment and, to a lesser extent, levodopa  reward over a delayed (larger) reward. However, the direction of the effect of dopamine on the reward system is inconsistent. For example, some studies showed lower levels of impulsivity on 1 and 2 mg/kg doses of hetamine , 18 wherhetamine –21 or nohetamine .d-amphetamine decreases impulsive behavior on the Stop task and in the Go/no Go task , and decreases delay discounting  and single-photon emission computed tomography (SPECT) can provide a direct measurement of putative dopaminergic differences between PD with and without ICDs. These nuclear medicine techniques use molecular imaging to assess biochemical, neurochemical, or pharmacological processes in the brain. For example, changes in neurotransmission can be detected using radiotracers with high affinity for dopamine receptors.2/3 post-synaptic receptors availability. The spatial resolution of current PET and SPECT machines allow separate assessment of the dorsal and ventral striatal regions, and their components .When a radiotracer is injected, it competes with dopamine for binding to free dopamine receptors. Thus, if dopamine is released endogenously, radiotracer binding can therefore be used as a marker for dopamine release . AccordiA limitation of the PET and SPECT studies of ICD in PD published so far is the small sample size, with the largest study including 21 PD patients with ICD and 68 without ICD  and the A meta-analytic approach can overcome these limitations. Low powered studies can be combined and differences in striatal dopaminergic function between PD patients with and without ICD estimated with a higher reliability. To the best of our knowledge, no previous meta-analysis has been published on this topic.2/3 receptors availability in the ventral and dorsal striatum.Therefore, the objective of this study was to investigate differences in dopaminergic function in the striatum in PD patients with and without ICD. To this aim, we systematically reviewed and meta-analyzed PET and SPECT based reports on dopamine transporter level, presynaptic dopamine release, and post-synaptic DThe PubMed, Science Direct, EBSCO, and ISI Web of Science databases were searched for peer-reviewed studies on PET or SPECT striatal dopaminergic function in PD-related ICD and published from database inception until the 7th of March 2018.The following search string was used: “[(Parkinson's disease OR Parkinson) AND ] AND (Positron emission tomography OR PET OR Single Photon Emission Computed Tomography OR SPECT OR SPET OR DaTSCAN).” A total of 384 papers were identified. After the exclusion of duplicates, 238 papers went through title and abstract screening. Two authors  independently screened titles and abstracts using Rayyan software  and 17 pStudies were included if they met the following inclusion criteria: (i) PET or SPECT study; (ii) PD patients without ICD (ICD–) compared with PD patients with ICD (ICD+); (iii) data reported for at least one striatal region; (iv) independence of the sample. Therefore, if a study sample was reported in multiple publications, only the study with the largest sample was included.We excluded reviews, case studies, commentaries, letters, abstracts and dissertations, conference papers, and postal surveys. Studies including drug naïve PD patients were excluded, as we were interested on ICD developed after dopamine replacement treatment (DRT) initiation. Moreover, drug naïve PD patients represent a different sample than those treated with DRT, as the former have shorter PD duration, and are not chronically exposed to DRT. Therefore, dopaminergic systems may be affected and stimulated differently in medicated and non-medicated PD patients.Studies in which PD patients underwent deep brain stimulation (DBS) were also excluded, as ICDs may either improve or develop after DBS . FinallyCorresponding authors of four studies , 34–36 wThe outcomes measures were the differences in the dopaminergic imaging parameters  between PD patients with and without ICD in striatal areas .I2 value with percentages around 25, 50, and 75 considered as low, moderate, and high, respectively , and dopamine receptors availability (postsynaptic) in the ventral striatum, dorsal striatum, putamen, and caudate. Data were analyzed using ReviewManager v5.3 . Standarectively . As PD sp < 0.05 was used as statistical significance threshold for all the analyses.Sensitivity analysis was performed by excluding studies clearly stating current antipsychotic or antidepressant use, as these drugs may affect dopamine receptor binding potential  or DAT uDemographic, clinical and methodological characteristics of the 117 ICD+ and 175 ICD– PD patients reported in the nine studies included in the meta-analysis are reported in Tables There was heterogeneity on the procedure to assess ICD across studies. ICDs were diagnosed either with a clinical interview based on the Diagnostic and Statistical Manual of Mental Disorders fourth edition text revision (DSM-IV-TR) , 44, 48,All patients were under DRT. In seven studies there were no between-group differences in total or dopamine agonist LEDD , 44, 48.One study divided the ICD+ group in single and multiple ICD subgroups . As the Six studies provided results in the left/right , 44, 48 n = 34, mean age = 32.5 years, SD = 8.8 years; mean, SD mm3 volume: putamen = 8805, 994; caudate = 5562, 865). SD was calculated accounting for the dependency of measures, by assuming a between-measures correlation of r = 0.5 in the striatal sub-regions. To test whether the whole dorsal striatum measure might have concealed differences in its sub-regions, analyses were repeated considering the putamen and caudate separately.Seven studies provided means and SDs for putamen and caudate separately , 44, 48.According to the radiotracer and the imaging approach used, studies were categorized as investigating (i) dopamine transporter level , 44, 48,In the dopamine transporter level subgroup, three studies , 34, 44 2/3 antagonist sensitive to changes in endogenous dopamine levels  and 175 were PD patients without ICD   but not in the ventral striatum . When dorsal striatum sub-regions were analyzed separately, both putamen  and caudate  tracer bindings were significantly reduced in the ICD+ vs. ICD– , putamen , and caudate . However, heterogeneity in the ventral striatum was high , putamen , and caudate , and heterogeneity .Heterogeneity was low for the dorsal striatum , but not in the dorsal striatum  and dorsal  striatal regions , dorsal striatum , putamen , and caudate , dorsal striatum , putamen , and caudate , dorsal striatum , putamen , and caudate , and heterogeneity .Sensitivity analysis was performed by excluding Payer et al. , which e2/3 receptors availability in the ventral and dorsal striatum. We found ICD+ to be associated with (i) lower DAT levels in the dorsal striatum and in its subdivisions  and (ii) reduced binding  in the ventral striatum in response to reward-related stimuli or gambling task, but (iii) no relationship between ICD+ and striatal post-synaptic receptors availability in either the dorsal or ventral striatum.This is the first systematic review and meta-analysis on PET/SPECT dopaminergic striatal correlates of ICD in PD. Our aim was to investigate if striatal dopaminergic function differs in PD patients with and without ICD. To this aim, we reviewed and analyzed studies on dopamine transporter levels, presynaptic dopamine release, and post-synaptic DICD+ group showed lower dorsal striatum DAT binding than the ICD– one.In the striatum, DAT is localized in axon varicosities and terminals that contain synaptic vesicles, as well as in non-synaptic region where it regulates and terminates extracellular dopamine activity . TherefoThe hypothesis of more severe degeneration of nigrostriatal projections in ICD+ patients is supported by a recent meta-analysis of case-control studies showing that the risk of ICD in PD increases with disease duration and being medicated for PD , two facThe lower DAT binding in ICD+ may also reflect medication-related DAT downregulation, but DAT regulation by DRT was found to be modest , 59. It ICD+ group showed reduced binding potential in ventral but not dorsal striatum when exposed to reward-related cues or when engaged in a gambling task.Participants to a gambling task are required to actively choose options associated either with reward or penalty and process related feedback. Conversely, in reward-related cues paradigms, participants passively view neutral or reward-related stimuli  without any active choice. Albeit being different, these tasks share neurobiological underpinnings. In pathological gamblers, reductions of ventral striatal and ventromedial prefrontal cortex activity have been documented in a gambling task  and rewa[11C]raclopride is sensitive to competition from endogenously released dopamine to a stimulus, therefore decreased binding potential found in ICD+ vs. ICD– groups in response to gambling tasks or rewarding stimuli reflects increased dopamine release. These findings are in keeping with functional imaging studies of behavioral and pharmacological addiction in the general population, whereby monetary and sexual stimuli elicit the same patterns of striatal activation as recreational drugs , 65. IncThese findings have important implications, since the exposure to any reward-related cue  may have the potential to increase abnormal dopamine release in vulnerable PD patients , as supp2-like presynaptic autoreceptors in the mesolimbic system reduces phasic dopamine release in the nucleus accumbens , and neuropsychiatric features  . The potOur meta-analysis showed specific patterns of dopaminergic dysfunction in the dorsal and ventral striatum in PD patients with ICD. These changes, which, to some extent, differ from those in people with ICD but no PD, may reflect either a preexisting neural trait vulnerability for impulsivity or the expression of a maladaptive synaptic plasticity under non-physiological dopaminergic stimulation .The study has been designed by AM, NE, and ST. Data have been gathered by AM and DDL, under the supervision of MS and ST. Data have been analyzed by AM. The manuscript has been drafted by AM, NE, and ST. AM, DDL, FL, MS, NE, and ST 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.


Molecular complexity is an important characteristic of organic molecules for drug discovery. How to calculate molecular complexity has been discussed in the scientific literature for decades. It was known from early on that the numbers of substructures that can be cut out of a molecular graph are of importance for this task. However, it was never realized that the cut-out substructures show self-similarity to the parent structures. A successive removal of one bond and one atom returns a series of fragments with decreasing size. Such a series shows self-similarity similar to fractal objects. Here we used the number of distinct fragments to calculate the fractal dimension of the molecule. The fractal dimension of a molecule is a new matter constant that incorporates all features that are currently known to be important for describing molecular complexity. Furthermore, this is the first work that reveals the fractal nature of organic molecules. A lot of the work mentioned below was triggered by the need to assess molecular complexity related to chemical synthesis. Molecular complexity is an intrinsic feature that depends only on the molecular structure but, nevertheless, provides an estimator for the synthetic effort2. Conversely, the accessibility of synthesis is a combination of intrinsic and extrinsic molecular properties as it depends on the applied educts, reagents and reaction conditions.Molecular complexity has been a topic of research for more than sixty years. Still, no common definition of the term ‘molecular complexity’ is currently available, even though it is a matter of daily discussions in synthetic organic chemistry laboratories. The lack of a common definition for molecular complexity recently resulted in a crowd-sourced approach3 aimed to calculate the information content of a living organism and, therefore, needed to know the information content of its integral parts such as organic molecules. Rashevsky suggested a formula to calculate information content of molecules, based on the Shannon entropy4. Rashevsky used terms ‘complexity’ and ‘information content’ synonymously. Decomposing a graph into its sub-graphs, while considering symmetry, was used by Mowshowitz for measuring graph complexity5. Based on the work of Rashevsky, Bonchev used the topological information content to calculate molecular graph features6. The topological complexity was based on the total number of connected subgraphs of the molecule7. As many other complexity measures, the topological complexity increases with the number of atoms and bonds. Independently from Rashevsky and Bonchev’s work, Bertz published a method to calculate a complexity index for a molecule8, also relying on the Shannon entropy. Bertz introduced the idea of using graph theoretical invariants for complexity calculations on chemical structures. A central term in his complexity considerations was the number of ways a subgraph can be cut out of a graph. In a more recent publication, Bertz and Herndon took the total number of subgraphs as measure for molecular complexity9. Hendrickson et al.10 later simplified the algorithm of Bertz and introduced correction for molecular symmetry. Randic introduced the ‘molecular connectivity index’ which he employed to calculate molecular complexity11. Based on the ideas of Bertz, Proudfoot has recently derived a molecular complexity measure that summed up the complexity of every atom environment in a molecule12.As pointed out above, molecular complexity naturally is a topic for chemistry; however, it originates in biology. In 1955, Rashevsky13, an algorithm later simplified by Borone and Chanon14. Similarly, Allu and Oprea15 used the number of chiral centers, added fused rings, functional groups, and electronegativity to calculate molecular complexity. A more general approach was developed by Rücker and Rücker16, utilizing a program to count walks on a molecular graph. Their work is also an example for a fluent transition between graph theoretical approaches and molecular feature based approaches.The domain-centered approach and the limited set of chemical elements and substructures used in organic chemistry, especially in medicinal chemistry, triggered the idea to develop another class of complexity measures for organic molecules by counting molecular features. Whitlock used the number of rings, unsaturated bonds, hetero atoms, and chiral centers to calculate the complexity of natural products18. It was shown by Sheridan et al. that some combinations of physico-chemical descriptors, such as the number of chiral centers, number of unique topological torsions, and the number of unique atom pair descriptors, correlated with the perception of molecular complexity by medicinal chemists1.Besides the efforts to describe molecular complexity with a single value, physico-chemical descriptors have been used for decades to obtain a number for molecular features like complexityHaving reviewed the available literature on molecular complexity, we identified two major approaches used to calculate molecular complexity.P(xi) being the number of graph features for an atom or a substructure xi. This reflects the increase in both entropy and complexity of the molecule with increasing number of information fragments. However, increasing the size of a molecule does not necessarily correlate with its increasing molecular complexity in the eyes of chemists.The first, graph theoretical approach evaluates substructure-related features and sums them up with the Shannon entropy formula that was initially developed to measure the content of binary information (1). For molecular complexity calculations, Rashevsky suggested using Shannon’s formula with The other approach uses weighting schemes to combine molecular features into a complexity number. A disadvantage of using weighting schemes is that it needs parameterization that can only be done with a limited number of molecules. A complexity calculation with a weighting scheme will only be valid for the classes of molecules the parameterization was done for.Intuitively, organic chemists agreed that certain features that bring variety into the molecular graph  also increase molecular complexity but only if these features are not repetitive. On the other hand, repetition of identical or very similar molecular features lowers the complexity of a molecule. Based on this point, we set out to calculate molecular complexity in a completely new way.During our work on repetitive molecular features we realized that every molecular substructure displays self-similarity to its parent structure. Self-similarity means that an object is similar to a part of itself. Let us derive the concept of self-similarity for organic molecules at the example of n-hexane, a linear alkane Fig. . A lineaA bit more structural variety is shown by heptanoic acid Fig. . For thiGlucose is an important biomolecule. Figure 19 and was established by Mandelbrot20 with the calculation of fractal dimensions. According to Mandelbrot, a fractal dimension is a ratio providing an index of complexity by comparing how a detail of a pattern changes with the scale at which it is measured21. An index of complexity is exactly what we desired to measure. From the manifold of existing algorithms to determine fractal dimensions we decided to calculate the fractal dimension of molecules analogously to the widely used Minkowski-Bouligand dimension (2)22.The obvious similarity of molecular subgraphs to their parent graphs triggered the idea to measure the self-similarity. Self-similarity is a well-established concept in mathematical and physical sciences. Measuring self-similarity started with Hausdorfε) versus log N(ε) for all ε. A common method to determine the Minkowski-Bouligand dimension is the box counting algorithm for two dimensional shapes. In the box counting algorithm, N equals the number of boxes that are needed to cover the shape for which the fractal dimension is determined. The scale ε is the side length of a box. With this scale, N boxes are needed to cover the shape. With a decreasing scale, ε increases the number of boxes N(ε) that are needed to cover the shape, i.e., both the nominator and the denominator of equation ). While the nominator stays unchanged, the number of features N that was defined as the number of boxes is now defines as the number of distinct subgraphs. The denominator can be simplified without changing the result.Thus, for the fractal dimension of a molecule the denominator of equation γmax versus log γmax, with γmax the number of bonds where the maximum number of distinct subgraphs occurred.Equation max). Moreover, glucose, the molecule from a commercial supplier with the PubChem Id 22578173 (PubChemCS) and strychnine displayed a strictly monotonic graph. The maximum, be it a single peak or a plateau in case of a molecule with repetitive structural elements (anandamide), was followed by a steep decline in N. This repeating pattern allowed calculation of the fractal dimension of each molecule using equation Figure Nmax and the corresponding bond count γmax. Instead of one distinct fragment, like in hexane, eleven distinct fragments were counted at γmax of eight bonds. Such low quantity of distinct fragments resulted in a small fractal dimension of 1.2.Adamantane is a highly regular alkane molecule, but in contrary to linear alkanes, it contains branches and bridged rings. Because of the bridged rings, it is impossible to apply the box-counting algorithm. So, the fractal dimension was only calculated by the maximum number of distinct fragments that could be cut out of the molecular graph. For all bond counts the number of distinct fragments was determined. The fractal dimension was calculated from the highest number of distinct fragments Anandamide consists of many more atoms and shows higher structural diversity than adamantane. It has also more atoms and bonds than glucose, nevertheless is the fractal dimension of glucose considerably higher. This finding comes without surprise, the number of distinct fragments in glucose is much higher than the number of distinct fragments in anandamide.max values.The molecule PubChemCS produced about 46,000 distinct subgraphs, which is almost three orders of magnitude above the number of distinct fragments for glucose. Hence, the fractal dimension of 3.5 for the PubChemCS molecule is just about one dimension above. This difference for the fractal dimensions of Glucose and the PubChem molecule is explained by the different bond counts at their γmax of 21 bonds. This resulted in the highest fractal dimension of 4.8 among all analyzed molecules in this study. The high fractal dimension of strychnine nicely reflects its synthetic complexity. The first total synthesis of strychnine by Woodward24 was awarded by the Nobel Prize in chemistry.Strychnine had the maximum of about two million distinct fragments at γIn contrast, the linear alkane molecule n-hexane from Fig. γmax in equation Here, we demonstrated for the first time that organic molecules show fractal properties as they can be subdivided into a number of subgraphs which show similarity to their parent graph. Because the fractal dimension of a molecule is clearly defined by the maximum number of distinct subgraphs at a certain bond count, the definition is even more robust than the original Minkowski-Bouligand dimension. For the calculation of the Minkowski-Bouligand dimension with the box-counting algorithm a number of scales is needed. However, this number has to be determined somehow, which introduces an additional degree of freedom into the calculation. For the calculation of the fractal dimension of molecules given in equation Organic molecules cover a range of five fractal dimensions. The simplest organic molecules, linear alkanes have a fractal dimension of zero. They contain self-similar substructures, with identical number of details for all bond counts. Therefore, the number of bond counts versus the number of substructures is a straight horizontal line. Another class of molecules is represented by anandamide. The number of substructures reached a wide plateau at bond count five. Such a plateau will be observed for fatty acids and other molecules containing long chains. Glucose and strychnine represent complex organic molecules. The shape of the curve has a steep ascent until it reached the maximum. So, from the shape of the curve different classes of molecular fractality can be identified. Further investigation will show how the curve shape can be applied to support molecule classification in drug discovery. The calculation of fractal dimension provides a new matter constant for chemical structures and a new tool for evaluation of how accessible these structures are for synthesis. Molecular complexity has been a point of discussion for decades by organic chemists. The fractal dimension of molecules is a complexity measure that encompasses all molecular features, does not depend on the molecular weight, and straightforward to implement.https://github.com/Actelion/openchemlib. Details for the calculation of the fractal dimension are in the example folder examples/FractalDimension/README.md. A jar file with the compiled sources can be obtained from https://mvnrepository.com/artifact/com.actelion.research/openchemlib.The complete Java code for all calculations is available on GitHub


This work presents an account of the reactivity behavior of the anticancer marine drugs, Soblidotin and Tasidotin, based on the calculation of the global and local descriptors resulting from Chemical Reactivity Theory (CRT), also known as Conceptual DFT, for their consideration as a useful complement to approximations based on Molecular Docking. The information on the global and local reactivity descriptors of the Soblidotin and Tasidotin molecules, obtained through our proposed methodology, may be used for the design of new pharmaceutical analogs by relying on the chemical interactions between these peptides and their protein-type biological receptors. It can be concluded that the CRT approximation to the global and local chemical reactivity, based on the descriptors, can provide interesting information for the consideration of both molecules as potential therapeutic drugs. This is complemented by a study on Advanced Glycation Endproduct (AGE) inhibition, by comparison with the usual molecular systems considered for the task, as a re-purposing study. Finally, the bioactivity scores for Soblidotin and Tasidotin are predicted through an empirical procedure, based on comparison with molecular structures with well-known pharmacological properties. There is considerable interest in finding new molecular systems that could be a basis for the design of novel therapeutic drugs. For the accomplishment of this objective, researchers have carried out searches for these potential therapeutic molecules in nature, based on experience acquired in recent decades. The sea is one of those places in which searches are carried out, as it is inhabited by an enormous variety of animal, plant, and microbial life, which generates diverse molecules and remains relatively unexplored.Among the chemical species that can be found in marine life, peptides are one of the most important chemical groups. Peptides are molecules formed by the combination of natural amino acids and some of their variants, and represent ideal chemical species for the design of therapeutic drugs, since they possess interesting properties inherent to the molecular systems, ranging from small amino acids to large proteins, with which they are often compared.Soblidotin, also known as Auristatin PE, has been investigated due its potential anti-tumor effects  and is aWhen one is working with Computational Chemistry and Molecular Modeling, there is not a universal methodology that could be applied to the entire spectra of known or unknown molecular systems. As such, our own devised methodology cannot be considered useful for all systems. The workaround that researchers in this field have found relies on studying, with a particular methodology, different but related families of molecules, in order to see if the proposed methodology can be applied with the same degree of success to the different group of molecules, and that the results obtained with a particular family of molecules are good not just by chance or some kind of serendipity. Indeed, the larger the number of studies with different groups of molecules, the greater the validity of the used methodologies.Thus, the objective of this work is to perform a comparative study of the chemical reactivity of the Soblidotin and Tasidotin peptides, of marine origin, by resorting to Chemical Reactivity Theory (CRT) , in addiRather than resorting to the usual methodologies in Medicinal Chemistry, related to Molecular Docking, QSAR, and QSPR, we prefer to study the potential usefulness of the peptides by considering Computational Chemistry and Molecular Modelling, based on CRT and some semiempirical relationships relying on collected databases.As with our recent studies ,7,8,9,10The molecular structures of the Soblidotin and Tasidotin peptides, which are depicted in The calculation of the maximum wavelength absorption of the Soblidotin and Tasidotin peptides was performed by conducting ground-state determinations with the same model chemistry. These results, together with the total, HOMO, and LUMO electronic energies, as well as the HOMO–LUMO gap, are shown in To get a glimpse of the electronic distribution around the atoms of both molecules, graphical sketches of the total electronic densities for Soblidotin and Tasidotin are presented in The definitions for the chemical reactivity descriptors, coming from CRT, are provided in terms of the energies of the HOMO and LUMO, 2(ϵL+ϵH) ,24, the ≈(ϵL−ϵH) ,24, the 4(ϵL−ϵH) , the eleϵL)2/16η , the eleϵL)2/16η , and theω±=ω++ω− . The resWe can see, from N, and If we now focus on the local reactivity descriptors coming from CRT, then the definitions will be: Function ,24, f−, which are considered to be the main reasons for the development of some diseases, such as Diabetes, Alzheimers, and Parkinsons .Many compounds have been devised as drugs with the goal of inhibiting the formation of AGEs, which include Pyridoxamine, Aminoguanidine, Carnosine, Metformin, Pioglitazone, and Tenilsetam ,35. PrevIf we consider that this relationship is valid, then, based on the calculated global descriptors for Soblidotin and Tasidotin, it should be possible to predict their AGE inhibition ability, by comparison with the mentioned drugs:It can be seen that the studied peptides possess AGE inhibitor abilities, which are lower than that of other molecular systems already on the market ,35.When looking for the potential therapeutic capacities of new molecules, an interesting approach is an empirical procedure based on comparison with the properties of molecular structures with well-known pharmacological properties. These values are known as Bioactivity Scores, and can be easily estimated by resorting to the free online Molinspiration software, as shown in The results from The outcomes of this work have resulted in the determination of important information about the chemical reactivity of two peptides of marine origin with potential therapeutic properties as anticancer drugs, Soblidotin and Tasidotin. This has been done by resorting to Chemical Reactivity Theory and some empirical relationships for the prediction of the pKa values, for their AGE inhibition abilities, and for the prediction of their Bioactivity Scores (as a measure of bioactivity).The total electronic densities for both peptides have been displayed in the form of graphical sketches, as well as the HOMO and LUMO orbitals, as an approximation to the electrophilic and nucleophilic Fukui functions.All these computed values could be of interest in the design and development of new pharmaceutical drugs, as well as for the practice of Medicinal Chemistry.


E-beam irradiation stimulates dynamics of fullerene molecules, which results in the first-order like structural transformation from the molecular crystal to molecular liquid. The real-time tracking of individual molecules using an automatic molecular identification process elucidates the relaxation behavior of a stretched exponential functional form. Moreover, the directly observed heterogeneous dynamics bear similarity to the dynamical heterogeneity in supercooled liquids near the glass transition. Fullerenes on graphene can serve as a new model system, which allows investigation of molecular dynamics in disordered phases.Structural rearrangements govern the various properties of disordered systems and visualization of these dynamical processes can provide critical information on structural deformation and phase transformation of the systems. However, direct imaging of individual atoms or molecules in a disordered state is quite challenging. Here, we prepare a model molecular system of C Understanding the structural changes in disordered non-equilibrium materials is important for their processing, yet it remains experimentally challenging to follow the dynamics. Here, Choe et al. image the first-order like transition from crystal to liquid in a model molecular system adsorbed on graphene by tracking individual molecules in real time. Various glassy materials are widely used in industrial applications and consumer products, and a fundamental understanding of the disordered non-equilibrium structural phase may also facilitate advancement in material processing and fabrication. Structural rearrangements, which are often spatially heterogeneous, govern the various properties of glassy systems6. Yet, direct visualization of these dynamical processes has been difficult, and diffraction-based analysis such as structure factors or intermediate scattering functions has been mainly utilized only to yield the spatially averaged signals2. To complement this issue, researchers have utilized computer simulations7 or other macroscopic model systems such as granular8 or colloidal particles11, providing verification on spatially heterogeneous dynamics and increasing characteristic length scales near the glass transition.Disordered states of materials, such as supercooled liquid or glass, display peculiar non-equilibrium behavior and have been intensively investigated for decades13, 2D carbon glass14, and Si atoms at the surface of amorphous Si15 have been successfully visualized using transmission electron microscopy (TEM) or scanning tunneling microscopy (STM). Although direct imaging of atomic structure has been successfully performed in these atomic disordered systems, in-depth observation and analysis of both structure and dynamics at the atomic or molecular resolution are still mainly lacking. In particular, the strong covalent bond in the previously-studied atomic glass leads to relatively slow structural evolution and dynamics under experimental conditions, limiting systematic studies on the dynamical behavior.The main experimental limitation in directly imaging atoms and molecules in disordered states can be overcome by modifying the sample geometry, to an atomically thin two-dimensional (2D) glassy system. Until now, 2D silica glass70 molecular system on graphene, and directly visualize both the structural and dynamical evolution of the system at molecular resolution. The relatively weak van der Waals interaction between C70 molecules can be perturbed using an electron-beam (e-beam) during aberration-corrected transmission electron microscopy (acTEM), emulating the melting process of the molecular crystal. Our computerized method precisely identifies molecular positions in the disordered state, and the pair correlation functions of molecules clearly show the short-range liquid-like ordering. Time-dependent relaxation behaviors of the molecular structure are studied in-depth by van Hove correlation functions, which clearly shows the relationship between the local structure ordering and the dynamical behavior of the system. Real-time tracking of individual molecules also allows us to extract the spatially heterogeneous dynamics during the melting process. Our study demonstrates that fullerenes on graphene can serve as a new model system for investigation of super-cooled liquid and glass at molecular resolution.Here, we prepare a C19. During e-beam irradiation, energetic electrons can occasionally transfer significant momentum and energy to the fullerene molecules via elastic collisions, generating structural displacements and dynamical motions20. In particular, the weak van der Waals interaction between fullerene molecules can be easily perturbed with an e-beam, inducing strong dynamical behavior. Indeed, researchers have previously observed an e-beam induced molecular dynamics of C60 in one-dimensional confinement of carbon nanotube21. In our study, we chose C70 deposited on graphene as an ultra-thin model molecular system. Graphene has high electrical/thermal conductivity and can further reduce the structural damage to samples because of fast energy transfers24. The atomically flat surface of graphene facilitates the dynamical behavior of the molecular liquid under imaging conditions, allowing systematic study of its molecular structure and dynamics. Compared to C60 fullerene25, C70 has a slight anisotropy in its molecular shape, which may also contribute to the more pronounced molecular movement.Fullerenes have relatively high robustness to e-beam under TEM imaging conditions compared to other organic molecules and can serve as model molecules70 molecules onto a graphene membrane by thermal evaporation to make a thin film (see methods section for detailed information)18. Using electron diffraction, we find that the nearest neighbour distance between molecules in the initial C70 film was 1.07 nm without any sign of anisotropy in terms of C70 orientation 16. The film also exhibits a uniform and well-ordered structure  were used.We deposited C70 packing structure can be perturbed with e-beam irradiation , can be used to efficiently identify molecular positions, even in an area where the direct recognition of molecules is difficult due to multiple molecular overlaps. To identify molecular positions reliably, we devised an image processing scheme whereby circles with a predesignated radius r were automatically identified. To achieve this, we processed TEM images through a two-phase Mumford–Shah (MS2) model34 and calculated the probability density function at each image pixel, PDF, which is a parameter indicating the probability of molecular presence at a certain pixel location . ) as shown in the inset of Fig. g2(r), including the peak intensity and position, were well-captured by our identification process. The identification process was also validated with control images of amorphous carbon and computer-generated random noise  were also tested, but we find that MS2 model yielded the most reliable identification of C70 molecules.To validate our method, we first applied our image processing to simulated TEM images using a model with disordered molecules on graphene, as shown in Fig. 2 model and PDF calculations, experimentally obtained TEM images were processed (Supplementary Movies r = 0.5 and 1.0 nm and converged to unity in the range r > 2.0 nm. This observation clearly shows that the C70 molecular system hosts a liquid-like short-range ordering under e-beam irradiation. It is noticed that the peak positions in 2D PCF were slightly different from the expected positions (red lines) calculated from projection image using close-packed C70 fcc molecular assembly. In particular, the peak positions were slightly down-shifted, showing an apparent lattice contraction compared to the original C70 crystal due to 2D projection of 3D counter-part37. We note that the obtained PCF in Fig. 38 is potentially available to obtain a static molecular 3D PCF, but it is currently challenging to apply the technique to our dynamic molecular systems due to possible e-beam induced motions during extended experimental imaging time.With the application of the MS70 molecules can be studied using time-dependent correlation functions. Van Hove correlation functions (vHCF) were obtained usingThe detailed dynamical behaviors of Cg = G/ρ at the state where the disordered state was developed from the C70 crystal (stage 2). We note that the proper calculation of vHCF involves the overall drift correction of time-series TEM images, which is described in detail in Supplementary Fig. r~0.5 nm and r~1.0 nm show rapid decaying behaviors and converge to unity  at the different stages of melting. The relaxation behavior can be fitted using τ and β. The fitting functional form does a good job of capturing the experimental relaxation, resulting in approximately τ~10 s and β~0.6. The observed non-exponential decaying function with β < 1  is a strong indicator of the heterogeneous dynamical behavior of the system39.Figure ity Fig. . The dynτ becomes smaller as the local structural order parameter, PCF 1st peak, diminishes. In situations where the structural order parameter is high, the relaxation dynamics can be suppressed due to the higher energy barrier originated from the larger molecular coordination number, which can be obtained from The observed dynamical relaxation characteristics exhibited correlations with local structure ordering. As shown in Fig. 70 molecules. Figure 70 molecules at a relatively early stage of melting (stage 1), demonstrating that the structural and dynamical behaviors of molecular disordering display spatially heterogeneous process. The central area of Fig. 11.The heterogeneous dynamical molecular behaviors can be further directly accessed by visualization of molecular movement during melting. With the ability to track dynamics at molecular resolution, we visualized the 2D diffusional behaviors of C6. The time scale of the transition from sub-free to free diffusional behavior is ~10 s, which is consistent with the relaxation time scale observed in Fig. 11. Calculated α2 at the stage 1 shows a peak value of around 2 at t~10 s as shown in Fig. α2 feature showing a peak at the characteristic relaxation time scale was previously observed for the alpha-relaxation of various glass systems5. This supports that the structural rearrangements during melting display heterogeneous dynamics, and our molecular system bears some similarity to the dynamics observed in supercooled liquids near the glass transition6. We note that the effect of overall drift was eliminated by the drift correction during the post-imaging process as shown in Supplementary Fig. Detailed diffusional behaviors of molecules can be examined using the mean-squared displacement of molecules defined by 70 molecular system and analysed its molecular dynamics with single-molecule sensitivity. The pair correlations for C70 molecules were calculated using an automated molecular position identification process. The spatially heterogeneous dynamical behavior of these molecules bore the similarity to the dynamical heterogeneity observed in supercooled liquids near the glass transition. Considering the possibility of using various molecules with different anisotropy  and modifying interactions between molecules through surface/internal functionalization (C60F48 and M@C82)16, fullerenes on graphene can serve as a new model system for investigation of molecular glass and supercooled liquid, providing unprecedented real-space imaging of dynamical heterogeneity. Future work combining the e-beam irradiation with in situ heating will make it possible to reveal the details on the thermodynamic equilibrium and non-equilibrium properties of fullerene model systems.In conclusion, we observed an e-beam induced first-order transition-like crystal melting of C40. Twenty-five micrometer-thick copper foil was used as the synthesis substrate. CVD graphene was transferred to Quantifoil holey carbon grids via direct transfer18. C70 films were deposited onto graphene TEM grids by thermal evaporation. Before the thermal evaporation process, TEM grids were pre-annealed in the air at 200 °C for 30 min to minimize surface adsorbate on graphene. The C70 films with thicknesses ranging from 0.5 to 10 nm were deposited at the deposition rate of 0.05 Å s−1 under a vacuum pressure of 2 × 10−6 Torr. The graphene substrate was held at 110 °C during the deposition.Graphene was synthesized using chemical vapor deposition (CVD)4 e nm−2 s−1. TEM videos were recorded using the CamStudio program with a temporal resolution of around 0.2 s per frame. TEM image simulations were performed using MacTempas software with experimental imaging conditions. The simulation images were obtained at a defocus value of −13 nm.TEM imaging was performed using a FEI Titan equipped with an image aberration corrector operated at 80 kV and a JEOL ARM 200 F equipped with image and probe aberration correctors operated at 80 kV. Selected area electron diffraction was performed with a FEI Tecnai. The electron dose during TEM imaging was ~2 × 102) model and the calculation of the probability density function (PDF) for molecular presence at each pixel. First, the MS2 model calculates the support function of an experimental TEM image. Based on the dark circular shape of fullerenes on the image, the annulus support with a given radius was applied to each image pixel. Second, the PDF was computed using the calculated support function and the chosen reference molecule, which described the probable molecular positions. To find positions that otherwise could be missed due to the aggregation of molecules, bright artificial molecules were inserted to the positions obtained from PDF, after which the PDF was re-calculated. Double-counted positions were determined if the PDF value was more than 98% of the PDF maximum. Drift correction of time-series TEM images was performed using a custom ImageJ macro. To quantify overall drift for each frame, tracking of molecular positions was executed for all frames, and the average displacement of the tracked molecules at a given frame was calculated along both the x and y directions. The overall drift was compensated using the calculated average displacement for a given frame. These processes are described in greater detail in the Molecular position identification was performed using the two-phase Mumford–Shah (MSSupplementary InformationDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4


Kaposi's sarcoma (KS) is a kind of cancer that causes flat or raised lesions containing Human herpes virus 8 (HHV8). The KS lesions are common among immunosuppressed HIV patients. Highly Active Antiretroviral (HHART) treats and prevents the development of KS. The objective of this study was to determine the presence of K1 and K15  genes in Kaposi's sarcoma-associated herpes virus (KSHV) among immunosuppressed patients due to HIV-1.This was a cross-sectional descriptive study where consecutive sampling technique was adopted to pick archived tissue blocks from the Thematic Unit of Anatomic Pathology, Department of Human Pathology, College of Health Sciences, University of Nairobi and Department of Laboratory Medicine, Histology Section, Kenyatta National Hospital.Upon staining 81 tissue blocks with H & E, 84% (68/81) were diagnosed as KS and 16% (13/81) as KS-like. The K1 and K15 (P) genes were both detected at 88.9% (72/81) in the tissue blocks, with 95.8% (69/72) detection from KS and 4.2% (3/72) from the KS-like.The K1 and K15 (P) genes of KSHV were present among the immunosuppressed patients with Human Immunodeficiency Virus (HIV)-1. It is important to carry out K1 and K15 (P) genes detection on tissues that are diagnosed as KS or KS-like by histology technique. Current studies indicate that all types of Kaposi's sarcoma (KS) are caused by Human Herpes Virus (HHV)-8 infections, and the difference in them is attributed by the contributions of many cofactors . GeneticHypothesis: the existence KSHV gene K1 and K15 (P) gene is associated with KS among patients who had HIV-1 and AIDS.4 cell count, tumor location, number of tumors, and antiretroviral treatment status of the patients whose tissue blocks had been collected and used were obtained from the registry records in the cytology and human pathology department. For this study, the inclusion criteria were tissue blocks that were previously diagnosed as KS or KS-like with a comprehensive bio-data information linking the blocks and registry records. Tissue blocks previously diagnosed as KS or KS-like that had missing or incomplete bio-data information from the registry records were excluded from the study. Consecutive sampling technique was used to pick a total of 81 tissue blocks from the 104 FFPE because they had met the inclusion criteria. A rotary microtome was used to section FFPE blocks.This was a cross-sectional descriptive study where 104 formalin-fixed paraffin-embedded (FFPE) tissues blocks were retrieved from the Thematic Unit of Anatomic Pathology, Department of Human Pathology, College of Health Sciences, University of Nairobi and Department of Laboratory Medicine, Histology Section, Kenyatta National Hospital between the year 2013 and 2016. The demographics, HIV-1 status, CDTissue sectioning: different blades per FFPE block were used to prevent carry-over of DNA. After each section, the surface of the microtome was sterilized using DNAZapTM PCR DNA (Thermo Fisher Scientific Company Cat No./ID: AM9890) degradation solutions. One tissue section was cut up to 10 µm thick. The tissue sections were processed for Hematoxylin and Eosin (H and E) staining and the results reported by a qualified human pathologist.DNA extraction: DNA extraction was carried out from the sectioned tissues using Qiagen kit GeneRead DNA FFPE (Qiagen®company Cat No./ID: 180134).The extraction kit removes paraffin and reverses formalin cross-links from the DNA tissues before it is bound to the QIAampMinElute column. The eluted DNA was then ready to be used for nested PCR.DNA detection: taq PCR Core Kit-Qiagen (Qiagen® company Cat No./ID: 201223) was used to detect K1 and K5 (P) gene. The two (K1 & K15 (P)) selected genes of KSHV were detected using two set of primers. The set of primers used were; K1 product size 868 bpK1a-f ATGTTCCTGTATGTTGTCTGC; K1a-r AGTACCAATCCACTGGTTGCG K1 product size 840 bp K1b-f GTCTGCAGTCTGGCGGTTTGC; K1b-r CTGGTTGCGTATAGTCTTCCG, K15 (P) product size 365 bp K15P-OF TGCAGGCTTGGTCATGGGTTAC; K15P-OR GGGACCACGCTGCAATTAAATG; K15 (P) product size 285 bpK15-3C ACGCATACATGTACTGCCAC; K15-4C CTTTGATATTGCCAGTGGTG. The PCR cycling condition of all the two targeted KS regions were similar and it consisted of 30 number of cycles which entailed; initial denaturation at 94°C for 3 minutes, denaturation at 94°C for 1 minute, annealing at 63°C for 1 minute, extension at 72°C for 1 minute and final extension at 72°C for 10 minutes. The amplified PCR products were analyzed by electrophoresis on a 1% agarose gel containing ethidiumbromide (1µl/ml of agarose solution) and were visualized under ultraviolet light alongside 1Kb DNA ladder. A known case of KS was used as positive control and RNase free water used as a negative control.Ethical clearance: authors of this manuscript obtained ethical clearance for the study from Kenyatta National Hospital and University of Nairobi .Statistical analysis: sample size of 81 tissue blocks was calculated using the standard statistical formula [ formula t-test. A P value < 0.05 was considered statistically significant.where Z^2 = 1.96, p = 5.6% based on prevalence HIV infection, trends, and risk factors among persons aged 15-64 years in Kenya: results from a nationally representative study , q = 0.94 counts of the subjects whose tissue blocks were retrieved from the records demonstrated that none of the recruited subjects had a CD4 count of above 350 cell/ mm3. The number of CD4 count was grouped into two groups, 250 being the cut-off point. Among the tissue blocks that had the K1 and K15 (P) genes detected 51.4% (37/72) had CD4 count above 250 cells/ mm3 and 48.6% (35/72) had CD4 count below 250 cells/ mm3. The distribution of age and CD4 cell count were significantly skewed and were bimodal among those who had the K1 and K15 (P) genes detected at a P value of 0.039 and 0.004 respectively . The records also indicated the distribution and the number of KS lesions among these subjects whose tissue samples were archived as FFPE blocks. The number of KS lesions in these subjects was grouped into two, above 10 and below 10. The tissue blocks that the K1 and K15 (P) genes were detected and had > 10 KS lesions was 55.6% (40/72) and < 10 KS lesion 44.4% (32/72). The distribution of KS was grouped as either localized or generalized, with 37.5% (27/72) localized and 62.5 (45/72) generalized among those that the K1 and K15 (P) genes were detected. The distribution of the KS lesions among the tissue blocks that had the two targeted genes detected were as follows: skin 31.6% (23/72), lower limbs 27.8% (20/72), lymphedema 16.7% (12/72), upper limbs 13.9% (10/72), palate 5.6% (4/72), trunk 1.4% (1/72), genitals 1.4% (1/72) and eye 1.4% (1/72). The study did histology screening on the retrieved tissue blocks to describe the morphology of the KS lesions. Tissue blocks were processed, sectioned, stained by H & E, results read by qualified pathologist and were described as patches, plaques and/or nodules (The K1 and K15 (P) genes detected as positive were 88.9% (72/81) and negative was 11.1% (9/81). Among the 88.9% tissue bocks that were positive for the targeted HHV-8 genes, 41.7% (30/72) were from female and 58.3% (30/72) male. All the subjects whose tissue blocks were retrieved for this study were found to be HIV positive. The antiretroviral status of the subjects was also determined, and it showed that out of the 88.9% that had K1 and K15 (P) genes detected, 95.8% (69/72) were on antiretroviral treatment and 4.2 (3/72) were HAART naïve. The CD nodules . In this nodules  was done4 cell count, treatment and distribution of KS lesions showed a strength in the association: gender for example, we noticed that compared to females, males were twice represented among those with K1 and K5 (P) genes cases.Genotyping analysis in this study revealed that the Kaposi's sarcoma-associated herpes virus (KSHV) genes, K1 and K15 (P) were detected among the selected tissue blocks. These findings affirm the earlier hypothesis that K1 and K15 (P) genes of Kaposi's sarcoma-associated herpes virus (KSHV) were present in the tissue blocks retrieved from patients with HIV-1 and AIDS. A previous study  conducte3. Other studies [3 are linked to high risk of developing KS whether one is on HIV treatment or not. However, recommendations have been made on the need to lookout on KS cases in subjects with high CD4 cell count [4 cell count is elevated and clinical presentation of KS tumors either stabilize or resolve [These findings were in agreement with another study  where it studies , 25 havell count . For tre resolve . However resolve , althoug resolve . The mos resolve . Typical resolve . For thi resolve  and Zamb resolve  where it resolve  where woIn conclusion, Kaposi's sarcoma-associated herpes virus (KSHV) genes, K1 and K15 (P) were detected among tissue blocks collected from patients infected with HIV-1 and AIDs. The morphology of KS lesions upon histological diagnosis was distributed as patches, plaques and nodules. East Africa is traditionally known to have a high prevalence of KS . The preIn resources limited health facilities, Kaposi's sarcoma is routinely diagnosed by clinical presentations and cytological identification using H & E staining technique.There is need to detect HHV-8 using a robust diagnostic techniques to aid a precise patient management because the patients in this study had a dark skin pigmentation and in another study conducted on dark-skinned patients, KS had been confirmed to mimic a number of non-KS like dermatological conditions;It further stated that the patients who presented with violaceous skin lesions were likely to be considered a suspected case of KS which is difficult to demonstrate in dark-skinned individuals.The authors declare no competing interests.


A soluble epoxide hydrolase (sEH) mediates the metabolism of epoxy fatty acids to form the corresponding vicinal diols, which are usually inactive or less active than the epoxide substrates. The sEH enzyme presents in many organs, including but not limited to the liver, heart, spleen, lung, and kidney. Here we summarized the changes in the expression and activity of sEH in multiple renal diseases, such as acute kidney injury (AKI), diabetic nephrology (DN), chronic kidney diseases (CKD), hypertension-mediated renal damage, and other renal dysfunctions. We also discussed the pharmacologic effects and the underlying mechanisms of sEH inhibition by using an inhibitor of sEH and/or the generic deletion of sEH on multiple renal diseases. We believe that sEH is a potential therapeutic target for renal dysfunction although the target disease needs further investigation. EPHX2 (A soluble epoxide hydrolase (sEH), a member of epoxide hydrolases (EH) family, presents in almost all living organisms . In humaEPHX2 . The sEHEPHX2 . The epoEPHX2 .in vivo and in vitro. For example, EETs, the epoxide metabolites of ARA, are anti-inflammatory  with a high level in the renal proximal tubules but a low level in the renal distal tubules and meager presence in the glomeruli (n = 8) and those without known renal disorders (n = 7). The enzyme sEH was expressed in all the organs investigated including but not limited to liver, kidney, brain, stomach, intestines, pancreas, prostate, heart, lung, and skinlomeruli . In a folomeruli , 2018 alAlthough there has been no drug clinically used as a sEH inhibitor yet, a large amount of pre-clinically experimental evidence supports that sEH may be a potential therapeutic target for several kidney-associated diseases, such as acute kidney injury (AKI), chronic kidney disease (CKD), diabetic nephrology (DN), and hypertension-associated kidney damage.AKI is a common fatal disease in hospitals characterized by a sudden and sustained reduction in renal function with the phenotypes of an abrupt decrease in urine output and a dramatic increase in serum creatinine level. The mechanisms underlying the pathogenesis of AKI vary, including but not limited to ischemia/reperfusion, septic shock, toxicant exposure, and inflammation-caused decrease in kidney blood flow, resulting in the damage to renal tissues, and eventually renal dysfunction , which is.c.) injection of a sEH inhibitor n-butyl ester of 12-(3-adamantan-1-yl-ureido)-dodecanoic acid , odocytes .CKD is one of the top public health problems and leading diseases for all-cause mortality globally . The putin vivo and in vitro.Inhibition of sEH by both pharmacological interventions with a sEH inhibitor and target gene disruption of sEH has been reported to attenuate the renal injury in several murine and rodent models. cis-4-[4-(3-adamantan-1-yl-ureido)cyclohexyloxy]benzoic acid  attenuated the renal and cardiac injuries of the diseased rats by modification of the survival rate, blood pressure, cardiac hypertrophy, proteinuria, degree of glomerular and tubulointerstitial injury, and glomerular volume toward the normative status. In addition, c-AUCB with a standard renin-angiotensin system (RAS) blockade resulted in additional therapeutic effects in the improvement of rat survival rate, reduce in albuminuria, glomerular and tubulointerstitial injury when compared with the standard RAS blockade alone.By using a rodent model of CKD caused by 5/6 nephrectomy (5/6 NX) in Ren-2 transgenic rats, trans-4-[4-(3-adamantan-1-yl-ureido)cyclohexyloxy]benzoic acid  resulted in the similar fibro-protective and anti-inflammatory effects to sEH gene knockout  stress, inflammation, fibrosis, and autophagy toward normative conditions, which was further supported by the in vitro data from the pharmacological intervention of sEH with the sEH inhibitor TPPU  in the spontaneously hypertensive obese rats. In addition, co-administration of t-AUCB with rosiglitazone, an agonist of PPARγ, resulted in additive reno-protection , rs751141 (R287Q), and rs1042032, were reported to correlate with several renal-associated diseases. Based on current data, lower sEH activity is associated with the improved renal function. P < 0.001) and a lower sEH activity (P < 0.05) than those with the wildtype allele (G) (lele (G) . Also, Wlele (G) , 2018 reThe sEH has been reported to be upregulated by both endogenous and exogenous factors at both mRNA and protein levels. Ephx2 and chemical knockdown by various sEH inhibitors. In most cases, sEH is believed to be inhibited at protein level primarily due to the formation of the hydrogen bonds between the inhibitors and the active sites Tyr461 and Tyr385 in both human and mammal sEH enzymes  found that EETs protected the murine renal mesangial cells and tubular epithelial cells from palmitic acid-induced injury by activation of Ampk and Pax2, which was supported by the In addition to EETs, other epoxy fatty acids were also reported to contribute to the renoprotective role of sEH inhibition. 19(20)-epoxydocosapentaenoic acid, the principal epoxy metabolite of DHA that could also be stabilized by sEH inhibition, was found to mitigate the renal fibrosis in a mouse model of experimental UUO-induced renal fibrosis by decreasing renal epithelial-to-mesenchymal transition .EPHX1, has the similar function to sEH in mediating the hydrolysis of epoxy fatty acids , encoded by ty acids . Howeverty acids . Recentlty acids . The patN,N′-disubstituted substructure that is similar to many potent sEH inhibitors  and multiple doses (100–400 mg every 8 h) during the test period (1 week) and dose-dependent blood drug concentration and activity to inhibit the blood sEH with 90% or greater inhibition. This study suggested a twice-daily or thrice-daily dosing regimen for the patients . GSK2256In summary, sEH may be a promising therapeutic target for the prevention and treatment of renal disorders. Also, various small molecular sEH inhibitors were synthesized in several laboratories with favorable pharmacokinetics and pharmacodynamics in multiple animals and humans. To move sEH inhibitors toward clinical use, the next step should focus more on the functional investigation of sEH in the pathology and pathophysiology of the renal dysfunction by using human samples to target a renal disease for systemic evaluation of the pharmacological and toxicological effects of sEH inhibitors.J-YL designed and wrote the paper.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Z)-1-[4-(trifluoromethyl)benzylidene]thiosemicarbazide was successfully synthesized using thiosemicarbazide with 4-(trifluoromethyl)-benzaldehyde in ethanol solution. The data presented in this articles is related to our research articles entitled “Crystal Structure of (Z)-1-[4-(Trifluoromethyl)benzylidene]thiosemicarbazide”  [1]. This work shows the continue data from experimental spectroscopic measurement which are Fourier Transform Infrared (FTIR) and 13C Nuclear Magnetic Resonance (13C NMR). Assessment on the correlation with theoretical computational data was also carried out through GaussView 5.0.9 and Gaussian09 software. Molecular Electrostatic Potential (MEP) and Highest Occupied Molecular Orbital–Lowest Unoccupied Molecular Orbital (HOMO-LUMO) were also illustrated.New compound, namely ( Hence, the charge distributions can give the information about how the molecules interact with other molecules. In addition, determination the sites for electrophilic attack and nucleophilic reaction could be identified. Thus, the electrophilic reactivity is shown by the yellow regions and nucleophilic reactivity is shown by the blue region.MEP is useful to visualize variably charged regions of a (gap), hardness (η), softness (σ) and the global electronegativity (χ) were calculated by using Eqs. HOMO-LUMO determination and its several important key factors for conductivity activity which are the energy gap (ΔE