Fibrosis and hence capsule formation around the glaucoma implants are the main reasons for glaucoma implant failure. To address these issues, we designed a microfluidic meshwork and tested its biocompatibility in a rabbit eye model. The amount of fibrosis elicited by the microfluidic meshwork was compared to the amount elicited by the plate of conventional glaucoma drainage device.Six eyes from 3 New Zealand albino rabbits were randomized to receive either the novel microfluidic meshwork or a plate of Ahmed glaucoma valve model PF7 (AGV PF7). The flexible microfluidic implant was made from negative photoresist SU-8 by using micro-fabrication techniques. The overall size of the meshwork was 7 mm × 7 mm with a grid period of 100 μm. Both implants were placed in the subtenon space at the supratemporal quadrant in a standard fashion. There was no communication between the implants and the anterior chamber via a tube. All animal eyes were examined for signs of infection and implant erosion on days 1, 3, 7, and 14 and then monthly. Exenterations were performed in which the entire orbital contents were removed at 3 months. Histology slides of the implant and the surrounding tissues were prepared and stained with hematoxylin-eosin. Thickness of the fibrous capsules beneath the implants were measured and compared with paired student’s t-test between the two groups.P= 0.002). No complications were noticed in the 6 eyes, and both implants were tolerated well by all rabbits.The gross histological sections showed that nearly no capsule formed around the microfluidic meshwork in contrast to the thick capsule formed around the plate of AGV PF7. Thickness of the fibrotic capsules beneath the AGV PF7 plate from the 3 rabbit eyes was 90μm, 82μm, and 95 μm, respectively. The thickness at the bottom of fibrotic capsules around the new microfluidic implant were 1μm, 2μm, and 1μm, respectively. The difference in thickness of capsule between the two groups was significant (The microfluidic meshwork elicited minimal fibrosis and capsule formation after 3-months implantation in a rabbit model. This provides promising evidence to aid in future development of a new glaucoma drainage implant that will elicit minimal scar formation and provide better long-term surgical outcomes. Glaucoma is the leading cause of irreversible blindness in the world. To date,Trabeculectomy, glaucoma drainage implants (GDIs) and minimally invasive glaucoma surgeries (MIGS) are the currently available surgical treatment for glaucoma. Trabeculectomy is the standard surgical approach to treat adult primary glaucoma, but its success in children and certain glaucoma populations is limited.–7 GDIs wFig 1). The device design is based on that of the conventional GDI, however, we replaced the solid plate with a microfluidic meshwork in the fluid drainage region. The design of the meshwork was inspired by recently developed brain implants that can suppress chronic foreign body reactions.[Thus, there is a critical need to develop an implant that can sustain the aqueous outflow while preventing obstruction due to fibrosis–the key to long-term functionality. Here, we present the concept for a modified GDI . The microfluidic channels had outer diameters of 20 μm and inner diametersof 8μm. These parameters were determined according to finite element simulations to provide sufficient AH outflow (2 μL/min at 10 mmHg). After being released from the substrate, the meshworks were washed and stored in buffer solution prior to autoclave and implantation. The design, fabrication and simulation of the meshwork is documented elsewhere in details.[The drainage devices were fabricated using photolithography techniques similar to those that were demonstrated previously. The fabr details.Fig 3). The surgery was performed in standard fashion in the animal microsurgery suite at UCSF. Under an operating microscope, the rabbits were anesthetized using an intramuscular injection of a mixture of ketamine hydrochloride (50 mg/kg) and xylazine hydrochloride (10 mg/kg), followed by mask anesthesia of isoflurane (2–4%). All efforts were made to minimize suffering of the animals. Both eyes were then prepared with povidone-iodine. For each eye, 6–0 Vicryl suture was passed through the supratemporal limbus to rotate the eye downward. Conjunctival peritomy was performed at the limbus in the supratemporal quadrant, followed by posterior dissection in the same plane. The flexible microfluidic meshwork was placed without suture and the AGV FP7 plate was sutured with 9–0 nylon sutures onto the episcleral surface approximately 6 mm from the limbus. The conjunctiva was closed with interrupted 8–0 vicryl sutures. There was no communication with the anterior chamber with either implant. To facilitate visualization of the microfluidic meshwork, a limbal 10–0 nylon suture was placed at the middle of the microfluidic meshwork. At the end of the surgery, subconjunctival cefazolin 0.1ml was given for antimicrobial prophylaxis. As routine postoperative care, the rabbit eyes were treated with Polymyxin antibiotic drops for one week and prednisolone acetate 1% drops starting with 3 times a day and then tapered every 3 days. All animal eyes were examined for signs of infection and plate erosion on days 1, 3, 7, and 14 and monthly thereafter for up to 3 months. Three months after surgery, the rabbits were euthanized by intravenous injection of potassium chloride or sodium pentobarbital after being anesthetized by isoflurane or ketamine/xylazine combination.Three healthy Albino New Zealand rabbits  were purchased and maintained at the Laboratory Animal Resource Center (LARC) at the University of California, San Francisco (UCSF). The Institutional Animal Care and Use Committee (IACUC) of UCSF approved the study. All animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. For each rabbit, the eyes were randomized to have one assigned to the current AGV PF7 silicone plate (no tube connected) and the other to the microfluidic meshwork . The histological sections were examined and measured using light microscopy by a pathologist who was blinded to the different groups. Capsule thickness at the bottom of the plate was measured for each eye.Paired student’s t-test was applied to compare the difference in the thickness of fibrous capsule between the microfluidic meshwork and conventional AGV PF7 plate groups. The rate of infection/plate erosion and any other notable side effects were compared between the two groups using the Fisher exact test.Six eyes from 3 New Zealand rabbits underwent implantation of the plate of AGV PF7 in one eye and the microfluidic meshwork in the other in a randomized fashion. Two left eyes and one right eye received AGV PF7 while the other two right eyes and one left eye received the microfluidic meshwork. There was no tube connected to either AGV PF7 or the microfluidic meshwork. No significant complications were noticed during the implantation of both AGV PF7 and the microfluidic meshwork.Fig 4, we observed that a thick capsule had formed around the plate of the AGV PF7 while nearly no capsule formed around the microfluidic meshwork (brown). Average thickness of the fibrotic capsules beneath the AGV PF7 and the microfluidic meshworks were 89 ± 6.6μm and 1.3 ± 0.6μm, respectively. There was a significant difference between the two groups .During the postoperative visits, there were no signs of infections, inflammation or erosion in any eye. All rabbits tolerated both types of implants well. After 3 months, exenterations were performed and the entire orbits were processed for HE staining. As shown in Fig 6). This was not observed around monolayer meshwork.During further inspection of all the histological slides of meshwork implants, we noticed that some inflammatory cells accumulated in the region where the meshwork had stacked into multiple layers during surgery (< 10% of the total area, We presented here a modified concept of a GDI, in which the traditional plate of the GDI would be replaced by microfluidic meshwork. In this study, we tested our hypothesis that a properly designed meshwork can significantly suppress fibrotic tissue formation. In clear contrast to conventional implants, the microfluidic meshwork demonstrated excellent biocompatibility, evidenced by nearly no scar tissue and minimal inflammation.Scar formation is the key obstacle in the surgical management of glaucoma. Modifications to the surgical technique as well as addition of intraoperative and postoperative medications have been studied in an effort to modulate fibrosis and promote long-term success with variable results.–27 MitomThe most active phase of capsule formation occurs in the 3 months following implantation. AlthoughAdditionally, during the histological examination of the dissected specimens, we noticed some inflammatory cell activation at the edge of the meshwork implant wherever the meshwork was folded or stacked into layers during the surgery. These regions were less than 10% of the total meshwork area. We postulated that when the meshwork was folded into multiple layers, its flexibility and hence its biocompatibility with eye tissue was compromised. This led to increased inflammatory cell activation and aggregation. This was not noticed in the majority of the areas where a single layer was maintained.We chose a New Zealand rabbit model for this study because this model has been previously used to study the effects of various biomaterials as well as newly-designed glaucoma surgical devices on the degree of fibrosis.,37 SubseOne limitation of this study was that we only studied the tissue reactions to non-fluid draining implants. The mechanical mismatch between tissue and implant may not be the only factor affecting fibrosis. It is known that inflamed AH may also lead to inflammation around the implant and elicit tissue reactions, and thisIn summary, this study demonstrated that the flexible microfluidic meshwork elicited minimal scar formation after being implanted into rabbit eyes for 3 months. We believe that these encouraging results warrant further development of a new GDI based on the microfluidic meshwork. The resulting GDI may significantly reduce fibrosis around the device and improve long-term success rates of glaucoma surgery across the spectrum.


ADHD (attention deficit hyperactivity disorder) is a common neurodevelopmental disorder. There has been extensive clinical and basic research in the field of ADHD over the past 20 years, but the mechanisms underlying ADHD risk are multifactorial, complex and heterogeneous and, as yet, are poorly defined. In this review, we argue that one approach to address this challenge is to study well-defined disorders to provide insights into potential biological pathways that may be involved in idiopathic ADHD.To address this premise, we selected two neurogenetic conditions that are associated with significantly increased ADHD risk: Turner syndrome and the RASopathies (of which Noonan syndrome and neurofibromatosis type 1 are the best-defined with regard to ADHD-related phenotypes). These syndromes were chosen for two main reasons: first, because intellectual functioning is relatively preserved, and second, because they are strikingly phenotypically similar but are etiologically distinct. We review the cognitive, behavioural, neural and cellular phenotypes associated with these conditions and examine their relevance as a model for idiopathic ADHD.We conclude by discussing current and future opportunities in the clinical and basic research of these conditions, which, in turn, may shed light upon the biological pathways underlying idiopathic ADHD. ADHD  is currently defined in diagnostic manuals such as DSM-5  and ICD-Identifying and characterising the many dysfunctional biological pathways that culminate in the development of ADHD is a difficult, perhaps intractable, problem. On the one hand, studying children and adults with idiopathic ADHD is hindered by limited sample sizes and other issues that stem from within-group heterogeneity. On the other hand, it is difficult to model the complex cognitive and behavioural abnormalities seen in individuals with ADHD in animal models where experimental control is more feasible. The study of well-defined neurogenetic conditions in man circumvents, to some extent, challenges associated with etiological heterogeneity, whilst allowing the measurement of complex human behaviours in children and adult with these conditions.In this review, we examine the premise that human neurogenetic conditions that are associated with increased ADHD rates may help to provide converging evidence for biological pathways involved in the development of idiopathic ADHD. Specifically, we compare two phenotypically similar classes of human genetic conditions that may serve as such ‘experimental models’: Turner syndrome (TS) and the RASopathies. Of the known RASopathies, we focus on Noonan syndrome (NS) and neurofibromatosis type 1 (NF-1), conditions about which most data has been collated in the literature. Moreover, whilst ADHD symptomatology is reported across all of the RASopathies, including Costello syndrome, LEOPARD syndrome, and cardiofaciocutaneous syndrome (CFC), these disorders are associated with pervasive effects on global cognitive function that might confound more specific genetic effects on ADHD-related symptoms . TS, NF1Significantly increased ~18-fold) rates of ADHD have been reported in TS (~24%) relative to the general female population (1.3%) -fold rat, 10. ForThe presentation of ADHD-related symptoms can vary between TS, NF1 and NS. Girls with TS and ADHD present with pronounced hyperactivity (comparable to levels seen in idiopathic ADHD in boys)  and are An important question regarding ADHD-related phenotypes in these syndromes is whether they are the culmination of relatively discrete disruption to molecular, cellular and neural pathways, or whether they are simply a subset of the many adverse neurobehavioural consequences arising from gross brain maldevelopment; the fact that IQ is generally preserved across all three conditions indicates the former. Overall therefore, TS, NF1 and NS might be regarded as behavioural models of specific subtypes, or symptoms, of ADHD in the absence of general cognitive impairments. Currently, there is little clinical and neuropsychological information available that may indicate areas of commonality and difference across the three syndromes, and there is undoubtedly a need for further research in this area. Parallel in vivo and in vitro studies such as neuroimaging and animal/cellular model work may enable us to understand how similarities and differences in neuroanatomy, neurophysiology and neurochemistry across the three syndromes can contribute towards commonalities and dissociations in behavioural and cognitive phenotypes.In TS, although total brain volume appears comparable to that of healthy controls, there is evidence for decreased volumes of the cortex , hippocampus, thalamus and basal ganglia , 16. TheOngoing studies in mouse models with varying sex chromosomal complements are attempting to refine the brain sites that are most sensitive to X and Y chromosome dosage and that may therefore play a role in sex-biased cognitive constructs, including vulnerability to neurodevelopmental disorders such as autism and ADHD; interestingly, these appear to include systems previously implicated in ADHD vulnerability including the forebrain cholinergic system and cerebelo-pontine-thalamo-cortical circuitry . The X-mAt a structural level, the brains of subjects haploinsufficient for NF1 appear grossly normal, although hyperintensities, possibly reflecting aberrant myelin formations, are seen in patient MRI scans . In the KRAS mutation followed over the course of several years displayed epilepsy, and hippocampal sclerosis and atrophy [SHOC2 exhibit a relative megalencephaly, enlarged subarachnoid spaces, a relatively small posterior fossa, and a high rate of cerebellar tonsillar ectopia [The neurobiology of patients with NS has been studied to a limited extent (often in the context of a predisposition to cancer), and genotype-phenotype correlations have rarely been reported. Brain structure in NS appears grossly normal, but neurological abnormalities have been reported in some cases. Chiari I malformations have been noted in some cases of NS , whilst  atrophy ; neuroim ectopia .Neuropsychological differences between neurotypical 46,XX females and females with TS could potentially arise due to one or more fundamental genetic mechanisms.MAOA and HTR2C genes in phenotypes associated with ADHD [First, a plausible reason for increased ADHD rates in TS stems from the fact that these individuals, like males, are functionally hemizygous for some, or all, X-linked genes. Hence, the effects of X-linked mutations or polymorphisms that elicit ADHD-related phenotypes will be apparent, and will not be moderated by the effects of the second X-linked allele. Candidate gene studies have previously implicated polymorphisms within the X-linked ith ADHD , 42; recith ADHD ), is likThe second genetic mechanism that may explain why ADHD rates are higher in TS than healthy control subjects is X-linked gene dosage. The epigenetically mediated process of X-inactivation silences one of the two X chromosomes within a female cell to ensure dosage equivalence  to male cells where only one X chromosome is active . HoweverM) results in poorer social function, and a greater degree of behavioural inflexibility, than inheriting a single paternally inherited X chromosome . Neurotypical 46, XX females inherit one X chromosome from either parent, and therefore any deleterious effects of the maternally inherited X chromosome may be compensated for by the presence of the paternally inherited X chromosome. Impaired social function, aberrant behavioural inhibition and behavioural inflexibility is commonly seen in individuals with ADHD [P and XM karyotypes differ  [The final genetic mechanism that may explain the elevated ADHD rates in TS is the parental origin of the X chromosome . Brieflyith ADHD . As a lay depth) ; howevery depth) , 51.The genetic mechanisms above could potentially influence brain function directly. Alternatively, they could exert effects on neurodevelopment and brain function indirectly through affecting the development and function of other organs. TS is commonly associated with impaired ovarian formation (and hence reduced levels of systemic reproductive hormones); the condition is also linked to abnormalities in levels of circulating growth hormone and thyroid hormones . An impoSRY (which is expressed in dopaminergic neuron-rich brain regions and is duplicated in cases of ADHD), and NLGN4Y and PCDH11Y, which encode cell membrane molecules important in maintaining synapse integrity and cell-cell interactions respectively, and both of which have been implicated as candidate genes for neurodevelopmental disorders [Given the data above, which sex-linked genes may be regarded as candidates for influencing attentional phenotypes in TS, and which neural circuitry may they influence? Y-linked genes that may be of particular interest with respect to ADHD include the male-determining gene isorders . In TS tSTS and NLGN4X (the X-linked homologue of NLGN4X) as candidate genes. STS escapes X-inactivation in both humans and mouse [Sts [Sts gene, or mice in which the associated steroid sulfatase enzyme is inhibited, show attentional deficits and other phenotypes of relevance to ADHD [STS are associated with the inattentive symptom count in boys diagnosed with idiopathic ADHD [Deletion mapping strategies  have been useful for identifying genomic regions that may potentially underlie X-linked gene dosage effects on executive function and social cognition. Zinn and colleagues have shown that individuals in which a small portion of the short of the X chromosome was missing (Xp22.3) consistently demonstrate abnormal neurocognitive function, including across an assay of attention (TOVA) on which subjects with ADHD are impaired . Of the nd mouse  and consuse [Sts ; moreove to ADHD , 59, 60.hic ADHD , 62.EFHC2 gene in these phenotypes in both females with TS and healthy males [EFHC2 and these phenotypes within idiopathic ADHD therefore represents a sensible future avenue for research. To date, no X-linked genes whose expression is significantly influenced in a parent-of-origin dependent manner have been identified in human somatic tissues, including brain. Interestingly, however, the closest human orthologue of the genomically imprinted maternally expressed mouse gene Xlr3b identified in a mouse model of TS [FAM9B, is located at Xp22.3 adjacent to the interval for TS neurocognitive function indicated by deletion mapping. Whilst FAM9B is apparently not expressed in the brain, it is expressed in other reproductive tissues [FAM9B activity may partially explain the X-linked POE on behaviour seen in TS, and assessing whether this gene may play a role in ADHD-relevant phenotypes related to circulating androgens [Deletion mapping in TS has also revealed a small dosage-sensitive locus at Xp11.3 that is linked to impaired emotion recognition and increased orbitofrontal cortex grey matter and amygdala size ; follow-hy males , 65, althy males . There ihy males –70; testel of TS , FAM9B,  tissues  and is a tissues . Investindrogens  will alsp > 0.5). Alternatively, the two groups may have differed in this cognitive domain as a function of their (epi)genotype. Theoretically, the bimodal distribution might have arisen due to phenotypic differences between 45,XP and 45,XM subjects, although, overall, and consistent with previous data [STS (and inherent genetic polymorphisms) described above, we hypothesise that the two groups may be distinguished by their genotype at this locus, or by systematic levels of metabolites dependent upon enzyme function .The bimodal peak in attention problems within a sample of participants with TS reported recently  is intrious data , the autFEN1/ELOVL6 [GSK3B [BAIAP2 [DPP6 [MYT1L [An alternative method to deletion mapping for identifying candidate genes underlying ADHD-related phenotypes in TS is to compare gene expression in case samples with that of neurotypical controls. Comparing tissue expression between the two groups is difficult for a number of reasons including the unavailability of the most pertinent tissue (i.e. brain), and obtaining TS samples from donors with consistent ages, karyotypes, hormonal profiles, and treatment histories. A comparison of cell-free RNA levels from the amniotic fluid surrounding TS or euploid female foetuses, which circumvents some of the aforementioned problems, has hinted at dysregulation of the hematologic/immune system in TS and has highlighted a number of specific X-linked and autosomal candidate genes for TS phenotypes ; of thes1/ELOVL6 , GSK3B [6 [GSK3B  and BAIA [BAIAP2 ) and as  [BAIAP2 ; of the P2 [DPP6  and MYT16 [MYT1L . ClearlyNF1 mutation exhibited excessive Ras activity, enhanced ERK and synapsin I phosphorylation and increased GABA-mediated inhibition, and consequently specific deficits in long term potentiation of potential relevance to learning deficits [Several converging lines of evidence have implicated GABAergic dysfunction in NF1 cognitive pathology. A seminal series of studies focussing on the hippocampus showed that mice heterozygous for a null deficits , 81; thedeficits .Of particular relevance to this review, NF1-deficient mice exhibit attentional impairments in a stimulus detection task dependent upon prefrontal cortex function . The extNF1 in Drosophila is associated with locomotor hyperactivity (an ADHD-like phenotype) that could be ameliorated by the administration of methylphenidate [Work in animal and cellular models have also shown that disruption of the Ras signalling cascade has downstream effects on dopaminergic function that may partially explain the cognitive deficits associated with NF1. Most germane to the ‘attentional’ focus of this review, is the observation that NF1-deficient mice exhibit a deficit in non-selective and selective attentional function in the absence of hyperactivity , a phenohenidate .PTPN11 effects in the central nervous system. At the cellular level, these studies show that Shp2, encoded by the PTPN11 gene, reduces myelination of axons [Brain and behavioural phenotypes in NF1 patients may also be influenced by altered levels of circulating glucocorticoids. It has recently been shown that deletion of the NF1 gene in the adrenal cortex increases circulating levels of stress hormones (corticosterone/cortisol) in mouse models and patients . In indiof axons  and is aof axons . In the of axons . Behavioof axons , 97, witof axons .Given the overlap in clinical and neuropsychological features between Turner syndrome and the RASopathies , is there any evidence for a shared underlying pathophysiology? There are some emerging data from clinical and model studies suggesting that the phenotypes of NF1-deficient individuals may be dependent upon gender, e.g. female patients are more likely to require treatment for visual decline than male patients, whilst only male NF1 knockout mice appear to show spatial learning/memory deficits, increased Ras activity and dopaminergic abnormalities . These dNF1 deficient patients and/or rodents show subtly altered CNS neuronal morphology including shorter neurite lengths, smaller growth cone areas and attenuated survival , alteredEif2s3x, Utx and Ddx3x, none of which have previously been implicated in ADHD. To reiterate, the relevance of the 39,XO mouse model for understanding the molecular neuropathology of TS is debatable given the lack of X-escapees in mice relative to humans [Eif2s3x, Utx or Ddx3x as differentially expressed genes. Hence, we may have failed to detect true brain gene expression commonalities between TS and NF1 models , or there truly is little overlap on this measure between the two models. Addressing which of these two possibilities is correct could be done through more relevant comparisons e.g. comparing gene expression in matched rodent brain tissues, or in induced pluripotent stem cell (iPSC)-derived neuronal cells from individuals affected by one or other of the two conditions.Further clues as to the shared molecular pathology underlying ADHD phenotypes between TS and NF1 may come from a comparison of gene expression in the two relevant mouse models , or cell lines from these mutants (see above) or patients. A comparison of whole brain gene expression in 40,XX and 39,XO mice revealed only a very small number of significantly differentially expressed genes  includino humans . Gene exo humans  and kineo humans  but do nPtpn11 mutations to those seen in cases of NS, have revealed abnormal myelination patterns [Ptpn11 models and the NF1 knockout model and it will be interesting to see the extent to which these overlap. In contrast, one might also expect to find differing neural phenotypes in these models given the opposing effect of these mutations on neurotransmitter phenotypes. Ptpn11 mutation increases basal excitatory synaptic transmission in hippocampal neurons and has a role in postsynaptic glutamatergic neurons through enhanced trafficking of AMPA receptors and effect on LTP dependent learning and memory [NF1 mutation increases inhibitory synaptic transmission in hippocampal neurons, and has a role in presynaptic neurons through increased GABA release [Given the current paucity of data regarding the neuroanatomy of NS patients, work in model systems will be fundamental to understanding the neural correlates of the cognitive and attentional deficits seen in this group. Data from genetically engineered mouse models and hippocampal slices harbouring equivalent patterns , increaspatterns ; the LTPd memory . NF1 mut release , 81.Whilst we now know that ADHD rates are elevated in Turner syndrome and the RASopathies, we still have very little information on ADHD subtypes and co-morbidities in these populations, about how ADHD-related phenotypes vary with age in these conditions, and about their underlying psychological, neural and molecular substrates. Therefore, more detailed clinical, neuropsychological and neuroanatomical phenotyping across cases and appropriate neurotypical controls across development is required; there also should be an emphasis on measures of brain function  particularly during tasks taxing ADHD-related constructs such as alerting, orienting and executive control , with a c-fos immunohistochemistry or fMRI) during performance of such tasks, in TS and RASopathy models will enable high resolution of the aberrant neural circuitry underlying any cognitive phenotype; such predictions may then be tested experimentally through techniques such as optogenetics or DREADDs. Further work in iPSCs originating from TS or RASopathy patient samples, or their neuronal derivatives, will also provide clues as to molecular processes that may underlie ADHD-related phenotypes, and that could be followed up further in animal models and, ultimately, patients.The brains of patients with TS or a RASopathy cannot be studied intimately in vivo. Hence, useful animal and cellular models for these conditions have been, and will continue to be, critically important for understanding the basis of associated cognitive deficits. There are now a number of excellent behavioural assays available for mice that tax processes of relevance to ADHD, many of which are conceptually and functionally analogous to human tasks for maximal translational value , 107–109Studies explicitly designed to look for commonalities and dissociations in abnormal brain structure or molecular dysfunction across conditions and models with ADHD-like phenotypes may also be undertaken to identify and characterise pathways that may frequently be disturbed in ADHD. This approach has recently been taken with regard to neuroimaging phenotypes in 26 mouse models recapitulating features of autism spectrum conditions  , and couNew insights into the molecular mechanisms predisposing to ADHD in TS may soon come from genome-wide genetic screens in idiopathic ADHD. To date, genome-wide association studies, copy number variant studies and exome/whole genome sequencing studies in ADHD have been limited by low power, a particular concern where the aetiology of the disorder is thought to be multifactorial and due to a complex mix of common and rarer variants. Moreover, these studies have traditionally neglected the X chromosome for a variety of reasons. With more ADHD samples now available for genotyping, a growing recognition that the X chromosome is influential in neurocognitive phenotypes, and better statistical tools for dealing with X-linked data, X-linked genes that are robustly implicated in idiopathic ADHD (and therefore that may play a key role in ADHD risk within TS) may soon be identified. Large-scale genome-wide studies in well-characterised ADHD samples may also potentially implicate genes involved in the Ras signalling cascade, including those involved in NF1 and NS; through interrogation of the clinical phenotypes associated with each variant, a clearer picture of how the various gene mutations may affect cognition and behaviour could be drawn.


ADHD is the most common neuropsychiatric condition affecting individuals of all ages. Long‐term outcomes of affected individuals and association with severe comorbidities as SUD or conduct disorders are the main concern. Genetic associations have been extensively described. Multiple studies show that intronic variants harbored in the ADGRL3 (LPHN3) gene are associated with ADHD, especially associated with poor outcomes.ADHD (MTA), initiated as a 14‐month randomized clinical trial of 579 children diagnosed with DSM‐IV ADHD‐Combined Type (ADHD‐C), that transitioned to a 16‐year prospective observational follow‐up, and 289 classmates added at the 2‐year assessment to serve as a local normative comparison group (LNCG). Diagnostic evaluations at entry were based on the Diagnostic Interview Schedule for Children‐Parent (DISC‐P), which was repeated at several points over the years. For an add‐on genetic study, blood samples were collected from 232 in the MTA group and 139 in the LNCG.In this study, we evaluated this association in the Multimodal Treatment Study of children with MTA participants, 14.6% retained the DISC‐P diagnosis of ADHD‐C in adolescence. For 127 LNCG participants, 88.2% remained undiagnosed by the DISC‐P. We genotyped 15 polymorphic SNP markers harbored in the ADGRL3 gene, and compared allele frequencies for the 30 cases with continued diagnosis of ADHD‐C in adolescence to the other participants. Replication of the association of rs2345039 ADGRL3 variant was observed .For the 205 ADGRL3 variants in the extreme phenotype of continued diagnosis of ADHD‐C from childhood to adolescence provides additional support that the association of ADGRL3 and ADHD is not spurious. Exploring genetic effects in longitudinal cohorts, in which refined, age‐dependent phenotypes are documented, is crucial to understand the natural history of ADHD.The detection of susceptibility conferred by  The MTA study was initiated as a 14‐month randomized clinical trial of 579 children diagnosed with DSM‐IV ADHD‐Combined Type  that transitioned to a 16‐year prospective observational follow‐up, and 289 classmates added at the 2‐year assessment to serve as a local normative comparison group (LNCG). Outcomes at the end of the randomized clinical trial and at follow‐up in childhood and adolescence have been reported , and adolescence (6 and 8 years after baseline) have been reported  susceptibility haplotype variants rs2345039, rs6551665, and rs1947274 previously reported . Allelic discrimination real‐time PCR reactions were performed in a 384‐well‐plate format in duplicate for each individual sample according to manufacturer's instructions. Briefly, 20 ng of genomic DNA was mixed with 2.5 μL of 2× TaqMan® Universal PCR Master Mix and 0.25 μL of 20× SNP Genotyping Assay in a total volume of 5 μL per well. Assays were run in an ABI 7900HT Fast Real‐Time PCR System . Allele calling was determined by end‐point fluorescent signal analysis using the ABI's SDS2.3 software .Genomic DNA was extracted from whole blood using the QIAamp DNA Blood Maxi Kit . Samples were subjected to whole‐exome genotyping using the Illumina® , an integrated collection of analytic tools for managing, analyzing, and visualizing multifaceted genomic and phenotypic data. Parameters for excluding markers from analyses included: (1) deviations from the Hardy–Weinberg equilibrium with P < 0.0000002 (0.05/250.000 markers) in both cases and controls, (2) a genotype call rate <90%, and (3) the presence of both more or less than two alleles. Genotype and allelic frequencies were estimated by maximum likelihood. Data subsets for common and rare variants where established using a minor allele frequency (MAF) criterion of 1.0% that was estimated from the whole sample data. Linkage disequilibrium (LD) pruning using CHM was applied to the subset of common markers using as parameters a window size of 50 markers with a window increment of 50 and an r2 threshold of 0.5. With this pruned subset of common markers, matrices of identity by descent (IBD) and probability of identity were estimated. Those were used afterward for the mixed models analyses.GenomeStudio data were imported to SVS 7.6.7, Golden Helix'sm‐1 variants are controlled for. These recently emerged methods have been proven to be more powerful than existing methods  . The minor allele C at the marker rs2345039 was significant (P = 0.005) after corrections by multiple comparisons (PFDR = 0.04), which is the same marker reported to be significant in the original manuscript that described variants harbored in ADGRL3conferring susceptibility to ADHD by our group  and response to stimulant medication (Arcos‐Burgos and Muenke ADGRL3 susceptibility variants interact with haplotype variants harbored at chromosome 11q22 in the region of NCAM1, TTC12, ANKK1, and DRD2 genes (Jain et al. ADGRL3 variants identifies a subtype of the condition characterized by the presence of severe externalizing symptoms and consequently a complex prognosis of persistence and graveness.A large number of studies show that ADGRL3 variant rs2345039, previously reported to be associated with the ADHD susceptibility haplotype, predisposes to the development of a refined ADHD phenotype characterized by persistence of ADHD combined subtype symptoms into adolescence. The fact that we are detecting association of the ADGRL3 variants in an extreme phenotype based on outcome of 6 years after childhood diagnosis – and confirmed by a rigorous prospective follow‐up – provides additional evidence that the association with ADGRL3 not only is not spurious, but also is associated with a more severe form of ADHD.In this manuscript, we present additional evidence that the P‐value of 0.10 was selected as the threshold to declare significance. The reason for selecting this threshold lies in the demonstration that for a given P‐value, the probability of replicating a significant result, under certain assumptions, is smaller than expected. Or better, as described by Goodman (Goodman These findings may partially explain the heterogeneity of longitudinal course in ADHD that includes differential symptom reduction with age, comorbidity, and ultimately, overall long‐term physical and mental health. Limitations of this study include the use of a final small sample size for those with persistent phenotype for ADHD at 6 years of age. However, it is known that in small sample size cohorts, where a high number of markers have been genotyped, major fluctuations in the distribution of significant values might appear and, consequently, spurious associations might reach significance. To control for this problem, we corrected by multiple comparisons using FDR as described in the Methods section. Furthermore, given that the direction of association between ADHD susceptibility and LPHN3 variants is well known, a  Goodman : “It is Continued research on this genetic variant in samples followed into adulthood, and that include comprehensive assessment of symptoms and functioning, is warranted.The authors declare no conflict of interests.


One of the most serious developmental problems in children is low birth weight in the world today . Babies A high prevalence of psychiatric disorders was showed such as anxiety and depression during pregnancy and poor in middle-income countries . ConsequThe study population included all infants born LBW and weight between the end of 2012–2013 in Isfahan Province with their parents. LBW rates in the Isfahan Province was reported about 10%, therefore, the total sample consisted of 400 live births in the period mentioned. Of these, 200 babies with low birth weights and weight are desirable. Stratified according to the population in each city was conducted. In order to gather information on the properties of biological and environmental parental questionnaire for the first time .According to Mental, physical and emotional state of mother can affect the incidence of LBW. Finally, with increasing the quality of life and prevention of other risk factors, LBW can decrease.


The prevalence of obesity is increasing worldwide and prevention is needed. Whole grain has shown potential to lower the risk of obesity, cardiovascular disease and type 2 diabetes. One possible mechanism behind the benefits of whole grain is the gut fermentation of dietary fiber (DF), e.g. non-starch polysaccharides and resistant starch (RS), in whole grain. The purpose of the study is to investigate the effect of whole grain rye-based products on glucose- and appetite regulation.2 and subjective rating of appetite were measured the following morning at fasting and repeatedly up to 3.5 h after a standardized breakfast consisting of WWB. Ad libitum energy intake was determined at lunch, 14.5 h after evening test and reference meals, respectively.Twenty-one healthy subjects were provided four rye-based evening test meals in a crossover overnight study design. The test evening meals consisted of either whole grain rye flour bread (RFB) or a 1:1 ratio of whole grain rye flour and rye kernels bread (RFB/RKB), with or without added resistant starch (+RS). White wheat flour bread (WWB) was used as reference evening meal. Blood glucose, insulin, PYY, FFA, IL-6 as well as breath HP < 0.05) and increased the gut hormone PYY in plasma the following morning 0–120 min after the standardized breakfast, compared to WWB (P = 0.01). Moreover, RFB increased subjective satiety and decreased desire to eat, and both RFB and RFB/RKB decreased feeling of hunger (AUC 0–210 min). All rye-based evening meals decreased or tended to decrease fasting FFA  and increased breath hydrogen concentration . No effects were noted on energy intake at lunch or inflammatory marker IL-6 (0 + 180 min) after the rye-based evening meals, compared to WWB.The evening meal with RFB/RKB + RS decreased postprandial glucose- and insulin responses (iAUC) . Registered 15 January 2015.The study was registered at: ClinicalTrials.gov, register number NCT02347293 ( The worldwide increase in prevalence of obesity-related metabolic diseases such as the metabolic syndrome, cardiovascular disease and type 2 diabetes is predicted to continue . These dWG flour based products are usually rich in NSP, whereas the RS level is lower in comparison to WG kernel based products, making the total amounts of DF inferior in flour based products. It has previously been suggested that products made from WG flour with a low content of RS did not beneficially affect metabolic variables as oppose to WG kernel based products rich in RS. Consequently, opposite to what was observed after kernel based barley products, it was shown that barley flour porridge did not result in beneficial effects on glucose regulation in a 10.5–12.5 h perspective . In addi2) excretion was measured as a marker of gut fermentation, and subjective appetite ratings  were registered. Finally, ad libitum energy intake was registered at a lunch provided 3.5 h after the standardized breakfast.The aim of this study was to investigate the potential impact of WG rye products (bread), varying in botanical structure of rye and RS content, on cardiometabolic risk markers and appetite regulation, in a time perspective of 11–14.5 h after intake. For this purpose, four WG rye breads with different flour to kernel ratio and supplementation of commercial RS2 were provided to healthy subjects as evening meals. A white wheat bread was included as a reference evening meal. The rye-based test products and the reference product were consumed in the evening, in a randomized crossover study design, and test markers in blood were determined the next morning prior and following a standardized breakfast to measure glucose regulation (glucose and insulin), gut hormones (peptide YY (PYY)), blood lipids (free fatty acids (FFA)) and inflammatory tonus (interleukin 6 (IL-6)). Additionally, breath hydrogen . Approval of the study was given by the Regional Ethical Review Board in Lund, Sweden (Reference 2013/241).2). The inclusion criteria were age between 20 – 35 years, BMI between 19 and 25 kg/m2, non-smoker and no known metabolic disorders, food allergies or consumption of any specific diet (e.g. vegetarian or vegan diet). Recruitments of test subjects were ongoing between January 21 to March 3, 2015, and the experimental work was finished by May 22, 2015. Prior inclusion, each subject was given a full written as well as an oral explanation of the purpose and procedure of the study, and written informed consent was obtained from each subject. All test subjects were informed of the possibility of withdrawing from the study at any time they desired.Twenty-four healthy volunteers, 11 men and 13 women were enrolled in the study. Three of the subjects, 1 man and 2 women, did not complete all five visits. The twenty-one volunteers that completed the study and were included in the statistical evaluation were aged 25.3 ± 3.9 years (mean ± standard deviation (SD)), with normal body mass indices (BMI) (mean ± SD = 22.7 ± 2.3 kg/mRFB), 50% WG rye flour and 50% rye kernel based bread (RFB/RKB), 75% WG rye flour based bread with 25% added high-amylose maize flour  (RFB + RS), and 43% WG rye flour and 43% rye kernel based bread with 14% added Hi-Maize® 260 (RFB/RKB + RS). A 100% DM white wheat flour based bread (WWB) was included as reference product. The proportions of ingredients in the rye-based test products and reference product WWB are displayed in Table The study included four different rye-based test products, presented as cereal dry matter (DM): 100% whole grain (WG) rye flour based bread  and 6 g NaCl were mixed for 1 min in a food processor (Electrolux Assistent AKM3110W) and then 400 g lukewarm water was added and an additional 4 min of mixing was done. The dough was proofed at room temperature in a bread pan for 30 min. Baking was performed in a house-hold oven at 200 °C with steam the first 15 min and then the bread was covered with aluminum foil the last 45 min .300 g rye kernels were boiled in 250 g salted water (6 g NaCl) for 30 min and then set to cool in room temperature for 30 min. All water was absorbed into the kernels when cooked. 300 g whole grain rye flour and 6 g yeast  were added to the kernels and were mixed for 4 min in a food processor (Electrolux Assistent AKM3110W). The dough was proofed at room temperature in a bread pan for 30 min. Baking was performed in a house-hold oven at 200 °C with steam the first 15 min and then the bread was covered with aluminum foil the last 45 min .The bread was baked according to the same procedure as RFB, except that 200 g Hi-Maize was mixed together with the other dry ingredients .The bread was baked according to the same procedure as RFB/RKB, except that 100 g Hi-Maize was mixed together with the 300 g cooled rye kernels along with the other dry ingredients (300 g whole grain rye flour and 6 g yeast).After baking, the bread was cooled for 3 h, then placed in a plastic bag overnight. In the morning the bread was sliced into portions, wrapped in aluminum foil, put into plastic bags and stored in a freezer −20 °C). Portion sizes are presented in Table 0 °C. PorThe WWB was baked in a home baking machine ]) according to a standardized procedure. The bread ingredients consisted of 540 g white wheat flour, 360 g water, 4.8 g dry yeast and 4.8 g NaCl. After cooling, the crust was removed and the bread was sliced and portions were wrapped in aluminum foil, put into plastic bags and stored in a freezer (−20 °C). Portion sizes are presented in Table The test subjects stored the bread products in their freezer during the study period. At the day of consumption, they were instructed to take a portion from the freezer in the morning and thaw it at ambient temperature, still wrapped in aluminum foil and in the plastic bag. Optional amounts of water, coffee or tea (without milk and/or sugar) were allowed to be consumed with the test- and reference products.The standardized breakfast consisted of 122.0 g WWB, corresponding to 50 g available starch, and 200 ml of water. The breakfast bread was baked according the same standardized procedure as WWB, reference product.ad libitum energy intake, the test subjects were instructed to serve themselves with lunch consisted of fried Swedish hash, i.e. diced potato, meat and onion (Felix Krögarpytt Klassisk), from an oven sheet. The lunch was generously provided by Orkla Foods Sverige AB . According to previous analysis by Johansson et al. (2013) the nutritional content is 234 kcal/100 g of Swedish hash, whereof the proportion of macronutrients (% DM) is 38.5% carbohydrates, 13.9% protein and 27.5% fat 15], NSP 2 and VAS ratings for subjective appetite  were obtained at fasting (time 0 min) and at 15, 30, 45, 60, 90, 120, 180 and 210 min after commencing the breakfast. Blood glucose was determined on similar times points, excluding 210 min, and s-insulin was collected at all time points except 15 and 210 min. Plasma PYY was determined at time 0 min and at 120 min after breakfast. Serum IL-6 was obtained at time 0 min and at 180 min. Serum FFA was determined at time 0 min.Fasting and postprandial measurements were performed at all five visits. Finger-prick capillary blood samplings were applied for determination of blood glucose . Additional capillary blood samples were collected to determine serum (s-) insulin, s-FFA, s-IL-6 and plasma (p-) PYY. Breath hydrogen was measured as an indicator of gut fermentation activity, using an EC 60 gastrolyzer . Pre- and post-breakfast subjective appetite sensations  were obtained using a 100 mm Visual Analogue Scale (VAS). Breath HSerum insulin was determined with a solid phase two-site enzyme immunoassay kit , s-FFA concentrations with an enzymatic colorimetric method using a 96 well microplate . The quantitative determination of s-IL-6 was performed with an enzyme immunoassay  and the concentrations of PYY, both PYY (3–36) and PYY (1–36), in plasma were determined with a competitive enzyme immunoassay .Data are expressed as means ± SEM. Statistical evaluations involving glucose and insulin concentrations are based on incremental changes from fasting concentrations. The absolute values are used in the statistical calculations of the rest of the test variables. The incremental area- and area under the curve  were calculated for each subject and evening meal, using the trapezoid model. iAUC or AUC were used in the statistical evaluation of results regarding blood glucose, insulin and parameters for appetite sensations. Incremental peak (iPeak) concentrations were calculated for glucose and insulin as individual maximum postprandial increase from the baseline. GraphPad Prism  was used for graph plotting and calculation of AUC.P-values < 0.05. In the cases of unevenly distributed residuals (tested with Anderson-Darling and considered unevenly distributed when P < 0.05), a Box Cox transformation was performed on the data prior to the ANOVA analysis. Differences between the products at different time points during the experimental day were evaluated using a mixed model  with repeated measures and an autoregressive covariance structure. Randomization of the consumption order of test products was performed using a random number function in Microsoft Excel 2013 . If a result of a test variable from a test subject was missing for the reference product, the test subject was excluded from the statistical calculations of that specific test variable. If a result of a test variable from a test subject was missing for one of the test products, the test subject’s results for the remaining products will be included in the statistical calculations of that specific test variable. For breath H2, where the variation in the concentration changed scarcely over time, a weighted mean was produced by calculating a mean for equal time intervals (one mean per hour) over the test period, and then a mean for the different hours was calculated and used in the statistical analysis.Investigations with respect to significant differences in test variables depending on the different evening meals were assessed with ANOVA  followed by Dunnett’s multiple comparison procedure, using results after the reference product WWB as a control. The data was analyzed in MINITAB Statistical Software . The significance level was set at Primary outcome measure was change in blood glucose incremental area under the curve (iAUC) 0–120 min after the standardized breakfast. Number of participants required for the study was determined in MINITAB, using previous results from a study including single evening test meals consisting of rye kernels and the effects the following morning on glucose incremental area under the curve 0–120 min (iAUC) . AssuminP = 0.057), compared to WWB, see Table P = 0.100) in postprandial glucose responses (iAUC 0–120 min) to the standardized breakfast. There were no significant differences in fasting values after test products compared to reference product for neither glucose nor insulin concentrations.Compared to the WWB reference evening meal, the evening meal consisting of RFB/RKB + RS decreased iAUC 0–120 min) responses for glucose (−27%) and insulin (−21%) in the morning following the standardized breakfast, see Table 20 min reP < 0.05). Additionally, a tendency towards decreased FFA concentrations was shown after RFB/RKB .The fasting concentrations of FFA are presented in Table No differences were observed in the concentrations of IL-6 depending on the evening meal, see Table P < 0.05) as well as an increased postprandial mean concentration including time points 0 and 120 min .P-PYY response at the standardized breakfast is presented in Table 2 is presented in Table 2 levels at fasting, that remained increased after the standardized breakfast (P < 0.001).The response in breath HP < 0.01) and decreased feelings of hunger  and desire to eat  during the experimental day, 0–210 min after the standardized breakfast (AUC), compared to WWB. In addition, the evening meal RFB/RKB decreased the hunger sensation during the experimental day . No significant differences were seen in the energy intake at lunch depending on the evening meal.The data for subjective appetite ratings are shown in Table It has previously been shown that certain kernel based cereal products, e.g. barley kernel products, and more recently also rye kernel products, beneficially affected cardiometabolic test variables 11–16 h  after inThe hypothesis was confirmed, with respect to the RFB/RKB product with and without RS supplementation. Consequently, postprandial glucose regulation was improved and plasma PYY levels increased in a time perspective of 11–13 h after intake of the RFB/RKB + RS, compared to WWB. No such effects were seen after the RFB/RKB without RS supplementation. The results are thus in support of the importance of RS components, and/or the specific mixture of NSP and RS in kernels on metabolic regulation. However, independently of the RS content, all rye-based test products resulted in a decrease in fasting FFA concentrations the following morning compared to the WWB evening meal, with potential benefits on insulin sensitivity. An improved glucose regulation and decreased concentrations of FFA were recently observed in the morning when rye kernel based bread was included as an evening meal . Increas2 following the rye-based evening meals, the metabolic benefits seen in the present work are most likely emanating from gut fermentation. Substrate-induced increase in SCFA concentrations in the gut may potentially have beneficial implications on appetite- and metabolic regulation since SCFA has been suggested to stimulate the L-cells to secrete GLP-1 and PYY [ad libitum buffet served after 2 h, compared to infusion with saline [Previously, intake of rye kernels were shown to increase plasma levels of SCFA 10.5 h after intake [ and PYY , and in  and PYY . In the h saline , indicath saline . These gh saline  and coulDespite the significantly increased PYY levels in the present study, the effects on appetite variables and energy intake at lunch did not reach statistical significance after intake of RFB/RKB + RS. Instead, the non-supplement products, i.e. 100% flour based rye bread (RFB) and the RFB/RKB bread, which induced less increased PYY levels compared to RFB/RKB + RS, showed the most pronounced beneficial effects on subjective appetite ratings. In accordance, it has been reported that PYY levels do not always correlate with subjective appetite . Howeverad libitum breakfast [Although no significant effects on energy intake at lunch depending on the previous evening meals could be detected in this study, there was a non-significant decrease in energy intake after RFB/RKB evening meal (−7%) in comparison to the WWB. Consequently, this is in accordance with results after boiled rye kernels in a previous study with similar study design . A decrereakfast  or a brereakfast . In thisIt has previously been shown that an evening meal including WWB supplemented with a mix of RS (similar to the RS product used in the present study) and NSP (from barley) in proportions similar to the composition and content of indigestible carbohydrates in intrinsic barley kernels, significantly lowered the glucose response the following morning. Further it was observed that supplementation of a WWB with RS or DF separately did not improve glucose tolerance the next morning, compared to un-supplemented WWB , 9. ThisA limitation in the study was the small volumes of blood samples obtained, due to capillary blood sampling instead of venous blood sampling. Consequently, it was not possible to investigate additional parameters of inflammatory variables and gut hormones. Another potential study limitation was the relative low number of subjects and the low energy intake at breakfast when investigating subjective appetite sensations during the experimental day and voluntary food intake at lunch. Nevertheless, the appetite ratings and energy registration from the present study indicate that rye-based products, in particular the combination of WG rye flour and rye kernels (RFB/RKB), have the potential to lower energy intake at the subsequent day, but further studies are necessary to confirm this.The present study showed that addition of RS to a rye bread based on 50% intact kernels and 50% WG flour (DM) beneficially impact metabolic parameters in an 11–14 h perspective, as demonstrated by improved postprandial glucose regulation, decreased concentration of FFA at fasting and increased levels of PYY. In addition, it was shown that WG rye flour bread contributed to increased sensation of satiety and that WG rye flour bread solely or combined with rye kernels lowered perceived hunger sensations, 11–14.5 h after intake. The findings suggest that WG rye has anti-diabetic and anti-obesogenic potential. More studies are needed to further evaluate the metabolic effects of inclusion of commercially available RS fractions in different food matrices.


Flaviviruses are a group of diverse and emerging arboviruses and an immense global health problem. A number of flaviviruses are neurotropic, causing severe encephalitis and even death. Type I interferons (IFNs) are the first line of defense of the innate immune system against flavivirus infection. IFNs elicit the concerted action of numerous interferon-stimulated genes (ISGs) to restrict both virus infection and replication. Viperin  is an ISG with broad-spectrum antiviral activity against multiple flaviviruses in vitro. Its activity in vivo restricts neurotropic infections to specific regions of the central nervous system (CNS). However, the cell types in which viperin activity is required are unknown. Here we have examined both the regional and cell-type specificity of viperin in the defense against infection by several model neurotropic flaviviruses.−/− mice infected with Langat virus (LGTV). The effects of IFN pretreatment were tested in vitro in primary neural cultures from different brain regions in response to infection with tick-borne encephalitis virus (TBEV), West Nile virus (WNV), and Zika virus (ZIKV).Viral burden and IFN induction were analyzed in vivo in wild-type and viperinViperin activity restricted nonlethal LGTV infection in the spleen and the olfactory bulb following infection via a peripheral route. Viperin activity was also necessary to restrict LGTV replication in the olfactory bulb and the cerebrum following CNS infection, but not in the cerebellum. In vitro, viperin could restrict TBEV replication in primary cortical neurons, but not in the cerebellar granule cell neurons. Interferon-induced viperin was also very important in primary cortical neurons to control TBEV, WNV, and ZIKV.Our findings show that viperin restricts replication of neurotropic flaviviruses in the CNS in a region- and cell-type-specific manner. The most important sites of activity are the olfactory bulb and cerebrum. Activity within the cerebrum is required in the cortical neurons in order to restrict spread. This study exemplifies cell type and regional diversity of the IFN response within the CNS and shows the importance of a potent broad-spectrum antiviral ISG.The online version of this article (10.1186/s12974-018-1119-3) contains supplementary material, which is available to authorized users. The type I interferon (IFN) response is the first line of defense against virus infections. IFNs are induced and secreted in infected cells upon pathogen recognition. Secreted type I IFN binds to the IFN-α/β receptors (IFNARs) in both an autocrine and a paracrine manner, activating the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway, which induces the expression of hundreds of interferon-stimulated genes (ISGs) . ISG resFlaviviridae family such as hepatitis C virus (HCV), Zika virus (ZIKV), dengue virus (DENV), West Nile virus (WNV), and tick-borne encephalitis virus (TBEV) [Viperin  is an ISG with broad-spectrum antiviral activity . Viperins (TBEV) –21. Desps (TBEV) , 15, 18.s (TBEV) –26. Anots (TBEV) . They als (TBEV) , Langat s (TBEV)  and ZIKVs (TBEV) . Since tHere we have used LGTV as a model for tick-borne flavivirus infection in vivo. We found that viperin specifically restricted replication in vivo in the olfactory bulb and in the cerebrum, but not in the cerebellum. In primary cells isolated from the cerebrum, both neurons and astrocytes relied on viperin for their antiviral response. However, viperin failed to restrict highly pathogenic TBEV replication in the granule cell neurons from the cerebellum. Furthermore, we found that viperin was required for the IFN-mediated antiviral responses against flaviviruses in cortical neurons. In summary, our findings show that viperin acts as an inhibitor of flaviviruses in the brain with a region-specific role in the olfactory bulb and cerebrum and cell-specific role in cortical neurons.−/− mice on C57BL/6 background were a kind gift from Peter Cresswell, Department of Immunobiology, Yale University School of Medicine. In vivo infection experiments were performed at Umeå Center for Comparative Biology (UCCB) using LGTV strain TP21 . The virus was titrated by focus-forming assay on VeroB4 cells [C57BL/6 wild-type (WT) mice were purchased from Harlan laboratories/ENVIGO. ViperinB4 cells . Six- tod-lysine-coated T75 tissue culture flasks. Astrocytes were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS (Thermo Fisher Scientific), 0.2% penicillin and streptomycin (Gibco), and 2 mM L-glutamine (Gibco). Primary cortical neurons were isolated from the cerebral cortices of embryonic day 17, and cerebellar granule cell neurons from postnatal day 6 and 7 mice, and seeded onto poly-d-lysine-coated wells. Neurons were grown in neurobasal medium (Gibco) supplemented with B27 (Gibco), 0.2% penicillin and streptomycin, and 2 mM L-glutamine (Gibco). TBEV Hypr 71 (isolated in 1953 from patient blood in Czech Republic), LGTV TP21, and ZIKV MR 766 were kind gifts of Gerhard Dobler and were propagated in VeroB4 cells. JEV (Nakayama strain) and WNV  were kind gifts from S. Vene .VeroB4 cells were cultured in Medium 199/Earle’s Balanced Salt Solution (EBSS) (HyClone) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific) and 0.2% penicillin and streptomycin (Gibco). Primary mouse astrocytes and cortical and cerebellar granule cell neurons were isolated and cultured as described –32. In bTBEV, LGTV, and ZIKV were titrated using focus-forming assays as previously described , 29. In The amount of murine IFN-α in mouse serum was determined by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (PBL).Mouse tissues were homogenized in Trizol reagent (Invitrogen) using Lysis Matrix (Nordic Biolabs) and a Fast Prep-24 tissue homogenizer (MP). RNA was extracted using the Nucleo-Spin RNA II kit (Macherey Nagel). Five hundred nanograms of total RNA was used to synthesize cDNA with the QuantiTect Reverse Transcription Kit (Qiagen). Expression levels of mouse GAPDH, IFN-β, IFN-α2, Mx1, IFIT1, and CXCL10 were detected by validated QuantiTect primer assays (Qiagen) and the KAPA SYBR FAST (Kapa Biosystems) quantitative polymerase chain reaction (qPCR) kit using a StepOnePlus fast real-time PCR system as previously described . TBEV anAstrocytes and cortical neurons were treated with 5000 U/ml IFNαB/D  16 h befCells were lysed, and samples were prepared for SDS-PAGE as described . Samplest test was used to analyze qPCR data, Mann-Whitney test for in vivo experiments, and log-rank (Mantel-Cox) for survival analyses.Data from cell culture experiments were analyzed using Graphpad Prism software. Unpaired −/− mice were infected intraperitoneally (ip) with 104 FFU LGTV and bodyweight and survival was monitored. All mice survived the infections and did not display any overt signs of disease or loss in body weight  with a low (10 FFU) or a high (100 FFU) dose. At the low dose, all WT mice survived, whereas 60% of the viperinion Fig. . Howeverice Fig. . Thus, vtem Fig. –f. These, we next wanted to determine the role of viperin in response to highly pathogenic TBEV infection (Biosafety level 3). To identify the brain cell types in which viperin functions to restrict the spread of the virus through the cerebrum, we used primary cultures of neurons and astrocytes. First, the expression of viperin was analyzed after TBEV and LGTV infection in astrocytes and cortical neurons isolated from the cerebrum of WT mice. As a control, primary mouse embryonic fibroblasts (MEFs) were used in which the type I IFN response has been well characterized (39–42). Cell cultures were infected with either LGTV or TBEV (MOI 0.1) and analyzed at different time points post-infection for viperin RNA, protein expression, as well as for viral replication. All cell types were able to upregulate viperin upon LGTV and TBEV infection  were upregulated after IFN treatment of WT and viperin−/− cortical neurons  have shown that astrocytes within the olfactory bulb produce an IFN response, which restricts VSV infection. It induces long-distance IFN signaling within the brain, which activates antiviral ISG expression in distinct brain parts that prevents virus spread within the brain [−/− mice, LGTV disseminates into the cerebrum but not to the cerebellum in the absence of viperin. In addition, particularly cortical neurons rely on viperin to restrict viral growth in the presence of IFN, whereas granular cell neurons in the cerebellum can mediate an antiviral response in the absence of viperin. Our results indicate that the long-distance IFN signaling and antiviral response is blunted against LGTV in the absence of viperin.Few studies have sought to define the function of viperin in vivo. For WNV, higher viral burdens were observed in the spleen, kidney, and brain in the absence of viperin, which was evident as viperinnfection . Althoughe brain –45. We fThere are conflicting studies regarding the role of viperin in interferon production. One study showed that viperin enhances TLR-7 and TLR-9 signaling and facilitates the nuclear translocation of IRF-7 to induce type I IFN response in plasmacytoid dendritic cells (pDCs) , while a−/− mice. This was consistent with the observations that viperin inhibited LGTV in the olfactory bulb and cerebrum after peripheral viral replication. In WNV and VSV infection, an antiviral effect of IFIT2 has been observed in the olfactory bulb, cortex, cerebellum, and brain stem [−/− mice, regional restriction was observed in the brain stem and cerebellum in the context of peripheral viral infection, while only in the brain stem post-ic infection [Recent findings suggest differential immune responses within distinct regions of the brain that can modulate virus pathogenesis , 38, 49.ain stem , 51. Aftnfection , demonstAstrocytes are important producers of IFNs during neurotropic viral infections , 52. We Neurons are the main target of TBEV infection in the CNS. We have previously shown that both WT and in the absence of IPS-1 or IFNAR signaling, neurons are the main target of LGTV infection , 3. CortIn summary, we have found that viperin is important in specific regions of the brain to control virus replication. This dependency on viperin was further mapped to neuronal subtypes. We found that viperin was the most important ISG against TBEV and ZIKV in the cortical neurons, as IFN treatment could not compensate for the loss of viperin. Similar but not as dramatic effect was also found for WNV. These findings suggest that a single ISG can shape the susceptibility and immune response within different regions of the brain.Additional file 1:Figure S1. Intraperitoneal LGTV infection does not alter the body weight of WT or viperin−/− mice. 6- to 8-week-old WT and viperin−/− mice were inoculated with 104 FFU of LGTV by intraperitoneal route (n = 10). Body weight was measured daily for 21 days. (PDF 503 kb)Additional file 2:Figure S2. Viperin is highly upregulated in astrocytes after TBEV infection, and it inhibits LGTV infection in cortical astrocytes and neurons. Primary cortical neurons and cortical astrocytes were isolated and differentiated from WT mice and infected with TBEV . (A) Viperin expression was measured by qPCR at the indicated time points. Viperin mRNA was normalized to the GAPDH expression and represented as dCt. Primary WT and viperin−/− astrocytes (B) and cortical neurons (C) were infected with LGTV , and viral growth was determined at indicated time points by focus-forming assay. Statistical significance was calculated using unpaired t test, significance is indicated by asterisks . Data are cumulative from at least two independent experiments performed in triplicates. (PDF 541 kb)Additional file 3:Figure S3. Viperin is not needed for the induction of ISGs in cortical neurons and astrocytes. Primary WT and viperin−/− mouse cortical neurons and astrocytes were treated with 5000 U/mL IFNαB/D for 16 h or left untreated. mRNA expression of Mx1, IFIT1, and CXCL10 was determined by qPCR. Expression levels were normalized to the endogenous GAPDH expression and depicted as mRNA arbitrary units (AU). (PDF 531 kb)


BRCA1-like tumors correlate with improved response to DNA double-strand break-inducing therapy. A gene expression-based classifier was developed to distinguish between BRCA1-like and non-BRCA1-like tumors. We hypothesized that these tumors may also be more sensitive to PARP inhibitors than standard treatments.Patients with BRCA1ness) was developed using a centroid model with 128 triple-negative breast cancer samples from the EU FP7 RATHER project. This BRCA1ness signature was then tested in HER2-negative patients (n = 116) from the I-SPY 2 TRIAL who received an oral PARP inhibitor veliparib in combination with carboplatin (V-C), or standard chemotherapy alone. We assessed the association between BRCA1ness and pathologic complete response in the V-C and control arms alone using Fisher’s exact test, and the relative performance between arms  using a logistic model and adjusting for hormone receptor status (HR).A diagnostic gene expression signature (BRCA1-like status. In the I-SPY 2 neoadjuvant setting the BRCA1ness signature associated significantly with response to V-C (p = 0.03), but not in the control arm (p = 0.45). We identified a significant interaction between BRCA1ness and V-C (p = 0.023) after correcting for HR.We developed a gene expression signature to identify BRCA1-like signature was successfully translated to an expression-based signature (BRC1Aness). In the I-SPY 2 neoadjuvant setting, we determined that the BRCA1ness signature is capable of predicting benefit of V-C added to standard chemotherapy compared to standard chemotherapy alone.A genomic-based NCT01042379.I-SPY 2 TRIAL beginning December 31, 2009: Neoadjuvant and Personalized Adaptive Novel Agents to Treat Breast Cancer (I-SPY 2), The online version of this article (doi:10.1186/s13058-017-0861-2) contains supplementary material, which is available to authorized users. Histological subtype information in breast cancer is clinically relevant for treatment purposes but insufficient to describe all tumor heterogeneity . The basBRCA1 mutations are found in around 12% of all TN breast cancers . In. In35]. BRCA1ness signature in this validation set as a specific biomarker of V-C response, gene expression data from 116 HER2-negative patients  were analyzed. A Customized Agilent 44 K array (Agendia) was used to evaluate the 77-gene signature BRCA1ness classification. The association between BRCA1ness classification and response in the V-C and control arms alone was assessed using Fisher’s exact test, and the relative performance between arms  using a logistic model. We included adjustment for hormone receptor status (HR/TN) and tumor size in our model. Our sample size is small, and thus statistical calculations  are descriptive rather than inferential. This analysis does not adjust for multiplicities of other biomarkers evaluated in the trial but outside this study.To assess the BRCA1ness signature using whole genome gene expression data. The signature has been developed on fresh frozen (FF) breast tumors that were categorized as either BRCA1-like or non-BRCA1-like using a DNA copy number MLPA-based classifier [BRCA1-like tumors with a high sensitivity/specificity rate.We developed a assifier , and endBRCA1-like and the remainder was assigned to the non-BRCA1-like class. We employed the gene expression profiles of the tumors, DLDA and the labels assigned by the MLPA-based classifier to train a classifier that distinguishes between the two classes. Using the ROC area under the curve model (AUC) as the performance criterion we identified a 77-gene signature that resulted in the highest performance (Table Forty-eight percent of the tumors 61/128) in the discovery cohort were classified as /128 in tBRCA1-like status as defined by MLPA were 96.7% and 73.1%, respectively. Using Ingenuity Pathway Analysis (Qiagen) to identify key biological processes associated with the 77 genes in the BRCA1ness 77-gene signature, we found cellular assembly and control and DNA replication, recombination and repair to be among the top associated pathways and functions . Fifty-five patients were classified as BRCA1ness. Fourteen percent of these patients were hormone receptor-positive (ERα/PR) and HER2-negative. The distribution of pathological complete response (pCR) rates among BRCA1ness and non-BRCA1ness groups is shown in Fig. BRCA1ness classification and patient response was seen in the V-C arm  but not in the control arm   was also observed. Although there is enrichment for TN samples in the BRCA1ness group in univariate analysis (Table p = 0.023)  Fig. . A signi23) Fig. .Fig. 4I-p = 0.038). We use the likelihood ratio test to formally demonstrate that the logistic regression with the addition of the HR (and tumor size) terms does not provide a better fit to the data. When the hormone receptor-positive BRCA1ness classified patients are added to the graduating TN subset, the OR associated with V-C is 4.03. This is comparable to that of the TN alone (OR = 4.04), while increasing the prevalence of ‘biomarker-positive’ predicted V-C sensitive patients by 8%.In addition we found that the interaction also remains significant when adjusting for tumor size and HR .Because of results in preclinical models, hibitors , 42–44. hibitors . PreviouCA1-like –20, 24. herapies , 23, 24.BRCA1ness tumor may be partially explained in this manner. Serine biosynthesis has been identified to be essential to tumorigenesis in estrogen receptor-negative breast cancer cell lines [BRCA1ness gene expression signature, suggesting that tumors having BRCA1-like features may have particular vulnerabilities to drugs that interfere with serine biosynthesis. It would be interesting to test whether high expression of genes involved in serine and glycine biosynthesis can confer sensitivity to drugs which interfere with this biosynthesis in breast cancer cell lines. In addition, we found that the signature is capable of predicting response to the PARP inhibitor veliparib in combination with carboplatin compared with a control treatment regimen.Pathway analysis reveals that the genes in this signature are associated closely with cell cycle and cancer networks. We also observed a significant association with serine and glycine biosynthesis pathways with the genes of the signature. This is of particular interest because it has been recently shown that aerobic glycolysis signaling can promote tumor growth in breast cancer cell lines that are TN , indicatll lines . We idenBRCA1ness signature shows promise for predicting response to V-C combination therapy relative to control. We focused on the experimental arm of the study that contains DNA-damaging agents because the BRCA1ness test is meant to identify patients that may derive substantial benefit from these agents. We observed a proportion of patients who were hormone receptor-positive that benefited from the V-C treatment. It is unlikely in a regular clinical setting that hormone receptor-positive patients would be tested for BRCA1ness, but our data indicate that these patients could derive benefit from specific tailored treatments like PARP inhibitors and/or platinum agents. Concurrently reported results studying carboplatin in TN breast cancer have indicated it may be difficult to translate the pCR rate to longer benefit such as recurrence-free survival (RFS) [BRCA1ness classifier in relation to long-term benefit. In the event that downsizing of the tumor is required to facilitate conversion from mastectomy to breast-conserving therapy, this classifier may already have value. If verified in a larger trial, this signature may contribute to the selection criteria of PARP inhibitor trials in the future.The sample size in the I-SPY 2 TRIAL is small, but our prespecified analysis suggests that the al (RFS) –50. It sAdditional file 1:BRCA1ness signature genes. (XLSX 40 kb)Additional file 2:I-SPY 2 Trial participating sites and institutional review boards (IRB). (XLSX 48 kb)


Research has indicated that, compared with the general population, the prevalence of offenders with ADHD in prison is high. The situation for offenders managed in the community by the Probation Service is unknown. This study aimed to bridge the gap in our knowledge by (1) surveying the awareness of probation staff about ADHD and (2) screening the rate of offenders with ADHD managed within the service. In the first study, a brief survey was circulated to offender managers working in 7 Probation Trusts in England and Wales asking them to estimate the prevalence of offenders with ADHD on their caseload, the presenting problems of these offenders and challenges to their management, and the training received on the treatment and management of offenders with ADHD. The survey had a return rate of 11%. Probation staff perceived that 7.6% of their caseload had ADHD and identified this group to have difficulties associated with neuropsychological dysfunction, lifestyle problems and compliance problems. They perceived that these problems hindered meaningful engagement with the service and rehabilitation. Challenges to their management were perceived to be due to both internal processes (motivation and engagement) and external processes (inadequate or inappropriate interventions). Few respondents had received training in the management of offenders with ADHD and most wanted more support. In the second study, a sub-sample of 88 offenders in one Probation Trust completed questionnaires to screen for DSM-IV ADHD in childhood and current symptoms. The screen found an estimated prevalence of 45.45% and 20.51% for childhood and adulthood ADHD respectively and these were strongly associated with functional impairment. Thus probation staff considerably underestimated the likely rate, suggesting there are high rates of under-detection and/or misdiagnosis among offenders with ADHD in their service. The results indicate that screening provisions are needed in probation settings, together with training for staff. Rates vary depending on the screening methods and diagnostic criteria used, but a general consensus from data reported in international studies suggests that around 30% of adult male offenders in the prison population have ADHD Whilst the evidence base is growing for those detained in custodial settings, little is known about what happens ‘beyond the gates’ when many move into the care of probation services in the community. The role of criminal justice services in supporting offenders with ADHD in England and Wales was discussed at a meeting of experts from health  and criminal justice agencies in 2009. It was concluded that ADHD has begun to be recognized by the courts, prison and police services. However, a clear gap was identified within the probation service, which is a key service for providing support and management in the offender pathway. We therefore aimed to bridge this gap in knowledge by (1) surveying the awareness of probation staff about ADHD and (2) screening the rate of probation service-users with ADHD. In study 1 we investigated awareness by circulating a brief survey to offender managers working in seven Probation Trusts in England and Wales. In study 2 we estimated the actual prevalence rate of ADHD and associated functional impairment among a sub-sample of offenders managed within one Probation Trust using a screening protocol. It was hypothesized that rates of ADHD would be higher than those reported in the general population and similar to those reported in other forensic settings (H1); and that those screening positive for ADHD would report higher rates of impairment both in childhood and in their current functioning (H2).2.2.1.2.1.1.All probation officers/offender managers (around 600) working across seven Probation Trusts in England and Wales were invited to complete the survey.2.1.2.A survey was designed to meet the specific aims of this study that asked offender managers to estimate the prevalence of offenders with ADHD on their caseload, comment on the presenting problems of these offenders and challenges to their management, and describe the training they had received on the treatment and management of offenders with ADHD (survey available from corresponding author).2.1.3.Agreement to conduct study 1 was obtained from senior management in the Probation Trusts and ethical approval was awarded by the Psychiatry, Nursing and Midwifery RESC committee, reference PNM-1112-12. Senior management within each Trust circulated an email to all staff inviting them to participate and attaching an Information Sheet describing the purpose of the survey and providing a link to an online version. In addition a hard copy was attached to the email for staff who preferred to download the survey and return it by post.2.2.2.2.1.A total of 108 male offenders managed in the community by the West Yorkshire Probation Trust and aged between 18 and 25 years participated in the study. The West Yorkshire Probation Trust is the fourth largest in England and Wales supervising around 12,000 cases at any one time, most of whom are in the community. The majority of service users are young men, thus females and/or those aged 26 or above were excluded from the study.2.2.2.Participants completed the Barkley Current and Childhood Symptoms Scales The Barkley Symptoms Scales also include an assessment of impairment that enquires about function in specific areas of life activities. Participants rate the frequency with which they have experienced functional problems over the previous six months on a four-point scale for ten domains including home life, work or occupation, social interactions, community activities, educational activities, dating or marital relationships, money management, driving, leisure or recreational activities and management of daily responsibilities. Each domain can be summed to generate a total impairment score that ranges between 0 and 30.In addition, the current probation status of the offender was recorded for categories of (1) offender in the community, (2) offender in approved premises/hostel and (3) persistent ‘prolific’ offender. The categories broadly represent low to high risk levels for re-offending and/or risk of harm towards others2.2.3.Agreement to conduct study 2 was obtained by senior management in the West Yorkshire Probation Trust and approved by the Probation Research Board for England and Wales. Following a brief session of training in their administration, the ADHD rating scales (both child and current) were administered by staff to all offenders on their caseload who met inclusion criteria and who attended a routine healthcare assessment over a 12 month period. Probation staff read items to participants with literacy problems but they were given no further assistance or direction to complete the questionnaires.3.3.1.The survey response rate was poor at around 11% (N=68 respondents).3.2.The average number of offenders per caseload was 37.7 (range = 2 to 113). Out of a total of 2,563 offenders on their caseloads, staff estimated that 206 had ADHD (7.6%).3.3.Responses to the open question “Describe the three main presenting problems for ADHD offenders” indicated three main groups of difficulties, the most common relating to neuropsychological dysfunction . These deficits were perceived to hinder meaningful engagement in rehabilitation  and attenuate progress, for example due to time-management problems, missed appointments, lack of prioritisation and/or comprehension problems.The second category of presenting problems related to social and interpersonal problems . Within this category probation staff described how core symptoms of ADHD can negatively influence social skills and interpersonal relationships, and suggested that ADHD offenders may be “pushed away” by positive role models and easily re-engage with criminal peers.The third category related to difficulties associated with adhering to rehabilitation plans and included problems with attendance, maintaining appropriate boundaries, and accepting instructions. Low levels of self-esteem and motivation were also indicated, which may have hindered progress in their rehabilitation.3.4.Responses to the open question “Describe the three main difficulties in managing ADHD offenders” indicated two dominant categories relating to (1) internal processes (motivation and engagement), and (2) external processes (inadequate or inappropriate interventions). Motivational and engagement problems were perceived to be the primary management difficulty. One respondent mentioned how persisting, challenging behaviours often lead to the abandonment of motivational techniques, and teaching offenders with ADHD constructive life skills was perceived to be challenging due to their difficulties with self-regulation and limitations in social perspective taking.External factors referred to concerns about the adequacy of treatment programmes for offenders with ADHD. They were perceived as not receiving appropriate medication, leading to them being difficult to manage both in prison and in the community. In particular, difficulties were experienced “keeping ADHD offenders on target”, with staff suggesting that high levels of tolerance were required for their successful management and progress.3.5.Around one-fifth (19.1%) had attended talks or presentations on ADHD and a small percentage (5.1%) had received some ADHD training. Almost three-quarters (73.5%) of respondents reported they did not have adequate support from mental health services to manage offenders with ADHD, and just over half (52.9%) believed that there should be specialist workers within the probation service for managing offenders with ADHD.3.6.Of the 108 screens received, 20 were incomplete and could not be used. Of the remaining 88 participants, 53 (60.2%) were managed in the community, 17 (19.3%) were in a probation hostel, and 18 (20.5%) were classified as high risk or persistent prolific offenders.Of this group, 40 (45.45%) screened positive for childhood ADHD, of whom 8 (20.00%) were of predominantly hyperactive/impulsive type, 7 (17.50%) predominantly inattentive type, and 25 (62.50%) combined type see . Ten par3.7.As the impairment total variables did not meet criteria for parametric testing, Mann-Whitney U tests were conducted to compare the total impairment scores obtained on the child and adult ADHD symptom scales. A positive screen in childhood was associated with greater childhood  and current impairment , both with large effect sizes and both remaining significant when a Bonferroni correction was made to account for the two analyses see .A similar pattern was found for current ADHD criteria which was associated with greater childhood  and current impairment . Again, both results showed large effect sizes and remained significant following the Bonferroni correction.4.This study is the first of its kind with a focus on the probation service and, as hypothesised, there was a significantly greater estimated rate of offenders with ADHD being managed in the community by probation services than reported in the general population. The rates obtained were generally consistent with those reported in prison studies e.g. The offender managers identified that this group of offenders were more impaired than their non-ADHD peers and perceived that this impairment hampered their ability to engage with the service and successful progress in their rehabilitation. Particular issues related to neuropsychological, lifestyle and compliance problems and, for some, risk may be inflated due to these barriers that hamper their rehabilitation process. The casework relationship between offender managers and the offenders they work with involves a dual relationship of care and control yet they seemed to feel ill-equipped to support the offenders with ADHD on their caseload, identifying a clear training need. Very few offender managers reported they had received any training in ADHD and most responded that they needed more support from mental health services. Many reported that staff with specialist training in this area would be useful. Given the likely proportion that one-quarter of their caseload has ADHD, specialist supervision may be a cost effective solution.The pharmacological treatment of offenders with ADHD may have profound effects for both patient care and societal gains. Lichtenstein and colleagues Another Swedish study conducted a randomised controlled trial investigating both symptom and functional outcomes of treating offenders with ADHD with stimulant medication in the prison setting A strength of the current study is that it is the first one (so far as we know) to report the rates and management needs of offenders with ADHD in probation services. This is an important topic as the recognition and management of these offenders has the potential to benefit both the individual and society. The survey was conducted in a cross-section of the probation service that spanned a broad geographical area across the UK but there was a very low return rate. Nevertheless, those participating in the screening study were representative of offenders presenting with a broad spectrum of risk of re-offending and risk of harm towards others. However, the screening protocol was administered in only one Probation Trust and twenty screens were incomplete and had to be excluded from the sample. The Probation Trusts did not systematically record the number of offenders approached for inclusion in the study; the sample size was small and only male which may have further limited generalisability. In common with many prevalence studies of ADHD that apply a screening protocol, this study used self-reported rating scales to screen for symptoms of ADHD, one of which asks the participant to retrospectively recall symptoms in childhood which may not be accurate 5.There is a consistent over-representation of offenders with ADHD presenting in custodial and community probation settings. With respect to the latter, this study has highlighted two important needs. Firstly, screening protocols and procedures are required in probation settings in order to assist offender managers to identify offenders who may be affected by persisting symptoms of ADHD. Unidentified symptoms are likely to impact on both the ability of the offender to use the support that is available and the ability of staff to tailor appropriate treatment and support. Secondly there is a clear training need to support staff in their management of a challenging group of offenders with a high rate of functional impairment who may have difficulty adhering to a community treatment protocol and who are likely to benefit from treatment. The evidence regarding the vulnerabilities of offenders with ADHD in the criminal justice system is growing. We now have the knowledge base and something needs to be done as the evidence is taking us beyond ‘food for thought’ and towards a call for action.