Axon-derived PACSIN1 binds to the Schwann cell survival receptor, LRP1, and transactivates TrkC to promote gliatrophic activities.

Schwann cells (SCs) undergo phenotypic transformation and then orchestrate nerve repair following PNS injury. The ligands and receptors that activate and sustain SC transformation remain incompletely understood. Proteins released by injured axons represent important candidates for activating the SC Repair Program. The low-density lipoprotein receptor-related protein-1 (LRP1) is acutely up-regulated in SCs in response to injury, activating c-Jun, and promoting SC survival. To identify novel LRP1 ligands released in PNS injury, we applied a discovery-based approach in which extracellular proteins in the injured nerve were captured using Fc-fusion proteins containing the ligand-binding motifs of LRP1 (CCR2 and CCR4). An intracellular neuron-specific protein, Protein Kinase C and Casein Kinase Substrate in Neurons (PACSIN1) was identified and validated as an LRP1 ligand. Recombinant PACSIN1 activated c-Jun and ERK1/2 in cultured SCs. Silencing Lrp1 or inhibiting the LRP1 cell-signaling co-receptor, the NMDA-R, blocked the effects of PACSIN1 on c-Jun and ERK1/2 phosphorylation. Intraneural injection of PACSIN1 into crush-injured sciatic nerves activated c-Jun in wild-type mice, but not in mice in which Lrp1 is conditionally deleted in SCs. Transcriptome profiling of SCs revealed that PACSIN1 mediates gene expression events consistent with transformation to the repair phenotype. PACSIN1 promoted SC migration and viability following the TNFα challenge. When Src family kinases were pharmacologically inhibited or the receptor tyrosine kinase, TrkC, was genetically silenced or pharmacologically inhibited, PACSIN1 failed to induce cell signaling and prevent SC death. Collectively, these studies demonstrate that PACSIN1 is a novel axon-derived LRP1 ligand that activates SC repair signaling by transactivating TrkC.

Characterization of a Cdc42 protein inhibitor and its use as a molecular probe.

Cdc42 plays important roles in cytoskeleton organization, cell cycle progression, signal transduction, and vesicle trafficking. Overactive Cdc42 has been implicated in the pathology of cancers, immune diseases, and neuronal disorders. Therefore, Cdc42 inhibitors would be useful in probing molecular pathways and could have therapeutic potential. Previous inhibitors have lacked selectivity and trended toward toxicity. We report here the characterization of a Cdc42-selective guanine nucleotide binding lead inhibitor that was identified by high throughput screening. A second active analog was identified via structure-activity relationship studies. The compounds demonstrated excellent selectivity with no inhibition toward Rho and Rac in the same GTPase family. Biochemical characterization showed that the compounds act as noncompetitive allosteric inhibitors. When tested in cellular assays, the lead compound inhibited Cdc42-related filopodia formation and cell migration. The lead compound was also used to clarify the involvement of Cdc42 in the Sin Nombre virus internalization and the signaling pathway of integrin VLA-4. Together, these data present the characterization of a novel Cdc42-selective allosteric inhibitor and a related analog, the use of which will facilitate drug development targeting Cdc42-related diseases and molecular pathway studies that involve GTPases.

A qualitative study of the transition to employment of former university students on the autism spectrum from Australia and New Zealand.

Many former university students on the autism spectrum have poor employment outcomes despite strengths, qualifications, and ability. This article presents findings from a qualitative study of 11 former university students on the spectrum (self-identified: 2 males, 7 females, 2 non-binary, 18-50+ years), and five significant others (2 mothers, 3 spouses), from Australia and New Zealand. We identified issues associated with better and poorer transition to employment experiences (poor mental health, lack of support, poor interview skills). The former students also indicated a belief that it was not the role of a university education to prepare students for employment. This belief may have influenced their discipline choices and attitude towards using career supports and university employability components. The participants who studied non-vocationally specific qualifications described more difficulty with conceptualising their transition to employment, developing an employment goal, and finding work. It was suggested that universities may need to provide comprehensive transition to employment programs that develop the 'soft skills' required for employment and offer work placements for both vocationally and non-vocationally oriented courses to help students build skills that increase their employability.

Jak/Stat3 signaling promotes somatic cell reprogramming by epigenetic regulation.

Although leukemia inhibitory factor (LIF) maintains the ground state pluripotency of mouse embryonic stem cells and induced pluripotent stem cells (iPSCs) by activating the Janus kinase/signal transducer and activator of transcription 3 (Jak/Stat3) pathway, the mechanism remained unclear. Stat3 has only been shown to promote complete reprogramming of epiblast and neural stem cells and partially reprogrammed cells (pre-iPSCs). We investigated if and how Jak/Stat3 activation promotes reprogramming of terminally differentiated mouse embryonic fibroblasts (MEFs). We demonstrated that activated Stat3 not only promotes but also is essential for the pluripotency establishment of MEFs during reprogramming. We further demonstrated that during this process, inhibiting Jak/Stat3 activity blocks demethylation of Oct4 and Nanog regulatory elements in induced cells, which are marked by suppressed endogenous pluripotent gene expression. These are correlated with the significant upregulation of DNA methyltransferase (Dnmt) 1 and histone deacetylases (HDACs) expression as well as the increased expression of lysine-specific histone demethylase 2 and methyl CpG binding protein 2. Inhibiting Jak/Stat3 also blocks the expression of Dnmt3L, which is correlated with the failure of retroviral transgene silencing. Furthermore, Dnmt or HDAC inhibitor but not overexpression of Nanog significantly rescues the reprogramming arrested by Jak/Stat3 inhibition or LIF deprivation. Finally, we demonstrated that LIF/Stat3 signal also represents the prerequisite for complete reprogramming of pre-iPSCs. We conclude that Jak/Stat3 activity plays a fundamental role to promote pluripotency establishment at the epigenetic level, by facilitating DNA demethylation/de novo methylation, and open-chromatin formation during late-stage reprogramming.

Fluorescent substrates for flow cytometric evaluation of efflux inhibition in ABCB1, ABCC1, and ABCG2 transporters.

ATP binding cassette (ABC) transmembrane efflux pumps such as P-glycoprotein (ABCB1), multidrug resistance protein 1 (ABCC1), and breast cancer resistance protein (ABCG2) play an important role in anticancer drug resistance. A large number of structurally and functionally diverse compounds act as substrates or modulators of these pumps. In vitro assessment of the affinity of drug candidates for multidrug resistance proteins is central to predict in vivo pharmacokinetics and drug-drug interactions. The objective of this study was to identify and characterize new substrates for these transporters. As part of a collaborative project with Life Technologies, 102 fluorescent probes were investigated in a flow cytometric screen of ABC transporters. The primary screen compared substrate efflux activity in parental cell lines with their corresponding highly expressing resistant counterparts. The fluorescent compound library included a range of excitation/emission profiles and required dual laser excitation as well as multiple fluorescence detection channels. A total of 31 substrates with active efflux in one or more pumps and practical fluorescence response ranges were identified and tested for interaction with eight known inhibitors. This screening approach provides an efficient tool for identification and characterization of new fluorescent substrates for ABCB1, ABCC1, and ABCG2.

Heterozygous disruption of Hic1 predisposes mice to a gender-dependent spectrum of malignant tumors.

The gene hypermethylated in cancer-1 (HIC1) encodes a zinc-finger transcription factor that belongs to a group of proteins known as the POZ family. HIC1 is hypermethylated and transcriptionally silent in several types of human cancer. Homozygous disruption of Hic1 impairs development and results in embryonic and perinatal lethality in mice. Here we show that mice disrupted in the germ line for only one allele of Hic1 develop many different spontaneous malignant tumors, including a predominance of epithelial cancers in males and lymphomas and sarcomas in females. The complete loss of Hic1 function in the heterozygous mice seems to involve dense methylation of the promoter of the remaining wild-type allele. We conclude that HIC1 is a candidate tumor-suppressor gene for which loss of function in both mouse and human cancers is associated only with epigenetic modifications.

A descriptive examination of the types of relationships formed between children with developmental disability and their closest peers in inclusive school settings.

BACKGROUND: One of the most commonly cited rationales for inclusive education is to enable the development of quality relationships with typically developing peers. Relatively few researchers have examined the features of the range of relationships that children with developmental disability form in inclusive school settings. METHOD: Interviews were conducted with 25 children with developmental disability, aged 5 and 12 years, their 3 closest peers, and parents and teachers to examine 6 types of relationships. RESULTS: Behaviours associated with general friendship and acquaintance were the most commonly reported. Few dyads reported high rates of behaviour associated with special treatment, helping, ignoring, or intimate best friend relationships. CONCLUSIONS: The relationships of the majority of dyads were characterised by friendship or acceptance, but evidence of more intimate relationships was limited. An important direction for future research is the examination of ways to encourage more intimate relationships.

Emotion regulation in children (ERiC): A protocol for a randomised clinical trial to evaluate the clinical and cost effectiveness of Mentalization Based Treatment (MBT) vs Treatment as Usual for school-age children with mixed emotional and behavioural difficulties.

BACKGROUND: The majority of children referred to Child and Adolescent Mental Health Services (CAMHS) in the UK will present with mixed emotional and behavioural difficulties, but most mental health treatments are developed for single disorders. There is a need for research on treatments that are helpful for these mixed difficulties, especially for school-age children. Emotion Regulation (ER) difficulties present across a wide range of mental health disorders and mentalizing may help with regulation. The ability to mentalize one's own experiences and those of others plays a key role in coping with stress, regulation of emotions, and the formation of stable relationships. Mentalization Based Therapy (MBT) is a well-evidenced therapy that aims to promote mentalization, which in turn increases ER capacities, leading to decreased emotional and behavioural difficulties. The aim of this study is to test the clinical- and cost-effectiveness of MBT compared to treatment as usual for school age children with emotional and behavioural difficulties. If effective, we hope this approach can become available to the growing number of children presenting to mental health services with a mix of emotional and behavioural difficulties. MATERIALS AND METHODS: Children referred to CAMHS aged 6-12 with mixed mental health problems (emotional and behavioural) as primary problem can take part with their parent/carers. Children will be randomly allocated to receive either MBT or treatment as usual (TAU) within the CAMHS clinic they have been referred to. MBT will be 6-8 sessions offered fortnightly and can flexibly include different family members. TAU is likely to include CBT, parenting groups, and/or children's social skills groups. Parent/carers and children will be asked to complete outcome assessments (questionnaires and tasks) online at the start of treatment, mid treatment (8 weeks), end of treatment (16 weeks) and at follow up (40 weeks). TRIAL REGISTRATION: Clinical trial registration: ISRCTN 11620914.

The targeted delivery of multicomponent cargos to cancer cells by nanoporous particle-supported lipid bilayers.

Encapsulation of drugs within nanocarriers that selectively target malignant cells promises to mitigate side effects of conventional chemotherapy and to enable delivery of the unique drug combinations needed for personalized medicine. To realize this potential, however, targeted nanocarriers must simultaneously overcome multiple challenges, including specificity, stability and a high capacity for disparate cargos. Here we report porous nanoparticle-supported lipid bilayers (protocells) that synergistically combine properties of liposomes and nanoporous particles. Protocells modified with a targeting peptide that binds to human hepatocellular carcinoma exhibit a 10,000-fold greater affinity for human hepatocellular carcinoma than for hepatocytes, endothelial cells or immune cells. Furthermore, protocells can be loaded with combinations of therapeutic (drugs, small interfering RNA and toxins) and diagnostic (quantum dots) agents and modified to promote endosomal escape and nuclear accumulation of selected cargos. The enormous capacity of the high-surface-area nanoporous core combined with the enhanced targeting efficacy enabled by the fluid supported lipid bilayer enable a single protocell loaded with a drug cocktail to kill a drug-resistant human hepatocellular carcinoma cell, representing a 10(6)-fold improvement over comparable liposomes.

High throughput flow cytometry based yeast two-hybrid array approach for large-scale analysis of protein-protein interactions.

The analysis of protein-protein interactions is a key focus of proteomics efforts. The yeast two-hybrid system (Y2H) has been the most commonly used method in genome-wide searches for protein interaction partners. However, the throughput of the current yeast two-hybrid array approach is hampered by the involvement of the time-consuming LacZ assay and/or the incompatibility of liquid handling automation due to the requirement for selection of colonies/diploids on agar plates. To facilitate large-scale Y2H assays, we report a novel array approach by coupling a GFP reporter based Y2H system with high throughput flow cytometry that enables the processing of a 96-well plate in as little as 3 min. In this approach, the yEGFP reporter has been established in both AH109 (MATa) and Y187 (MATα) reporter cells. It not only allows the generation of two copies of GFP reporter genes in diploid cells, but also allows the convenient determination of self-activators generated from both bait and prey constructs by flow cytometry. We demonstrate a Y2H array assay procedure that is carried out completely in liquid media in 96-well plates by mating bait and prey cells in liquid YPD media, selecting the diploids containing positive interaction pairs in selective media and analyzing the GFP reporter directly by flow cytometry. We have evaluated this flow cytometry based array procedure by showing that the interaction of the positive control pair P53/T is able to be reproducibly detected at 72 hr postmating compared with the negative control pairs. We conclude that our flow cytometry based yeast two-hybrid approach is robust, convenient, quantitative, and is amenable to large-scale analysis using liquid-handling automation.

Cluster cytometry for high-capacity bioanalysis.

Flow cytometry specializes in high-content measurements of cells and particles in suspension. Having long excelled in analytical throughput of single cells and particles, only recently with the advent of HyperCyt sampling technology, flow cytometry's multiexperiment throughput has begun to approach the point of practicality for efficiently analyzing hundreds-of-thousands of samples, the realm of high-throughput screening (HTS). To extend performance and automation compatibility, we built a HyperCyt-linked Cluster Cytometer platform, a network of flow cytometers for analyzing samples displayed in high-density, 1,536-well plate format. To assess the performance, we used cell- and microsphere-based HTS assays that had been well characterized in the previous studies. Experiments addressed important technical issues: challenges of small wells (assay volumes 10 µL or less, reagent mixing, cell and particle suspension), detecting and correcting for differences in performance of individual flow cytometers, and the ability to reanalyze a plate in the event of problems encountered during the primary analysis. Boosting sample throughput an additional fourfold, this platform is uniquely positioned to synergize with expanding suspension array and cell barcoding technologies in which as many as 100 experiments are performed in a single well or sample. As high-performance flow cytometers shrink in cost and size, cluster cytometry promises to become a practical, productive approach for HTS, and other large-scale investigations of biological complexity.

Low-Grade Fumarate Hydratase-Deficient Renal Cell Carcinoma in a 30-Year-Old Female.

Fumarate hydratase (FH)-deficient renal cell carcinoma (RCC) is a rare and clinically aggressive RCC subtype that is commonly associated with the hereditary leiomyomatosis and renal cell carcinoma syndrome. The diagnostic hallmark of FH-deficient RCC is a high-grade microscopic appearance with prominent inclusion-like eosinophilic nucleoli and perinucleolar halos. Herein we report a case of an FH-deficient RCC in a 30-year-old female that exhibited low-grade nuclei and abundant eosinophilic cytoplasm, reminiscent of the clinically more indolent succinate dehydrogenase-deficient RCC subtype and the newly described eintity, eosinophilic, solid and cystic RCC. This case illustrates that FH-deficient RCC can have a wide spectrum of microscopic appearances, including low-grade eosinophilic RCC. In addition, it highlights that a low threshold to perform the immunohistochemical stains for FH and S-(2-succino) cysteine is warranted in RCC cases with unusual and even low-grade eosinophilic morphology.

A Structured Cognitive Intervention Pathway as a decision-support tool for non-pharmacological interventions within a dementia care service (Innovative practice).

Individual cognitive interventions for Alzheimer's disease have been shown to be beneficial and cost effective when evaluated as sole interventions. However, there is a need for a systematic, person-centric, structured approach to guide non-pharmacological intervention selection based on disease stage, symptoms, outcome assessment, and individual requirements. Our Structured Cognitive Intervention Pathway aims to facilitate the selection of first-line, or subsequent, non-pharmacological management for people with Alzheimer's disease living at home and in elderly care facilities. We discuss the Pathway's conceptual basis and evaluation of implementation as a decision-support tool within a dementia care service in China.

Short report: Learning through iconic gesture in autism spectrum disorder.

BACKGROUND: Understanding and therefore recalling spoken messages, including narratives, can be challenging for children with autism. While observing gesture can benefit narrative recall in typically developing children, whether observing gesture facilitates narrative recall in children with autism is unclear. AIMS: This paper examines whether observing iconic gestures affects narrative recall in children with a diagnosis of autism. METHODS AND PROCEDURES: We first identified iconic gestures to be observed by participants in the main study. Once appropriate iconic gestures had been identified, children with autism watched one video narrative with iconic gestures and one without gestures. While watching the video narratives, children wore Tobii Pro Glasses-2 to track their eye-movements. After watching each narrative, children were asked questions about the narratives to assess recall. OUTCOMES: Iconic gestures significantly benefitted narrative recall in children with autism beyond watching no gestures, and eye-tracking results suggested gestures helped children focus on the narrator. CONCLUSIONS AND IMPLICATIONS: Through identifying appropriate iconic gestures and producing them alongside a verbal narrative, gestures may successfully enhance learning in children with autism.

How Does Participation in Formal Education or Learning for Older People Affect Wellbeing and Cognition? A Systematic Literature Review and Meta-Analysis.

Objective: To determine the effect of later-life formal education or learning on quality of life (QOL), wellbeing, mood, and cognition. Methods: A systematic literature review of interventional clinical trials and observational studies was conducted for adults aged ≥55 years who had undertaken formal education or learning programs. Outcome measures included physical activity, happiness, affective and behavioral symptoms, cognitive function, and QOL. Bias was assessed using funnel plots, Egger's test, and leave1out analysis. Results: From 32 studies identified, we showed qualitative increases in cognitive function, life satisfaction, and self-confidence associated with learning. A meta-analysis revealed a significant pooled mean difference in MMSE scores (0.40, 95% confidence intervals = [0.12, 0.67]). Although there was a low risk of publication bias there was a high risk of sampling bias. Conclusion: Participation in formal education or learning contributed to increased wellbeing, QOL, healthy cognitive function, self-dependency, and a sense of belonging in older adults.

Birth of parthenote mice directly from parthenogenetic embryonic stem cells.

Mammalian parthenogenetic embryos are not viable and die because of defects in placental development and genomic imprinting. Parthenogenetic ESCs (pESCs) derived from parthenogenetic embryos might advance regenerative medicine by avoiding immuno-rejection. However, previous reports suggest that pESCs may fail to differentiate and contribute to some organs in chimeras, including muscle and pancreas, and it remains unclear whether pESCs themselves can form all tissue types in the body. We found that derivation of pESCs is more efficient than of ESCs derived from fertilized embryos, in association with reduced mitogen-activated protein kinase signaling in parthenogenetic embryos and their inner cell mass outgrowth. Furthermore, in vitro culture modifies the expression of imprinted genes in pESCs, and these cells, being functionally indistinguishable from fertilized embryo-derived ESCs, can contribute to all organs in chimeras. Even more surprisingly, our study shows that live parthenote pups were produced from pESCs through tetraploid embryo complementation, which contributes to placenta development. This is the first demonstration that pESCs are capable of full-term development and can differentiate into all cell types and functional organs in the body.

Self-concept of children with intellectual disability in mainstream settings.

BACKGROUND: Positive self-concept is an important educational outcome for individuals with disability. METHOD: Perceived competence and acceptance of 17 children with intellectual disability, included in mainstream classes, were assessed using the Pictorial Scale of Perceived Competence and Social Acceptance for Young Children (PSPCSA) and compared with (a) independently rated academic work samples and measures of social status, and (b) measures of perceived competence and acceptance reported for other populations of individuals with and without disability. RESULTS: Perceived competence and acceptance were positive and comparable to previously reported results for individuals with disability and young children without disability. The children's perceived cognitive competence was not consistent with ratings of their work samples but their perceived peer acceptance was more consistent with peer ratings of social status. CONCLUSION: Children with intellectual disability remained positive at an age when self-concept is likely to be negatively impacted by comparisons with higher performing peers.

Perspectives of Former Students with ASD from Australia and New Zealand on Their University Experience.

The university experience of students with ASD was explored through a qualitative study of 11 former university students and six significant others from Australia and New Zealand. A range of key issues were identified including difficulties encountered when studying, reasons for completion and non-completion, supports used, and coping strategies used by the participants. Many switched to part-time to manage their poor mental health and/or executive function and most had slow rates of progress. Also, some felt they had made poor discipline choices. The participants offered suggestions for future students and for making universities more autism friendly, and the possible need for transition and more structured study supports was identified.

An On-Line Survey of University Students with Autism Spectrum Disorder in Australia and New Zealand: Characteristics, Support Satisfaction, and Advocacy.

An on-line survey of 102 (51 females; undergraduate and graduate) university students with ASD across Australia and New Zealand examined student characteristics and satisfaction with academic and non-academic supports. A broad range of disciplines were studied, and the participants' reported strengths included a passion for learning, strong technology skills, and creative thoughts. The participants' greatest concerns were academic requirements and mental health, including high rates of self-harm and suicidal ideation. Despite support satisfaction ratings being high, support usage was low, possibly indicating a mismatch of supports and needs, lack of awareness of available supports, and/or poor advocacy skills.

Management of cognitive decline in Alzheimer's disease using a non-pharmacological intervention program: A case report.

INTRODUCTION: In China, the over 60 population is estimated to grow from 12% in 2010 to 33% of the overall population by 2050. The escalation in the aging population is projected to result in an Alzheimer's disease prevalence of 27.7 million people in China by 2050 causing substantial health and economic burden. While there are some published studies on multicomponent, non-pharmacological interventions for people with dementia, we have found no published community-based approach to care that encompasses personalized selection of non-pharmacological interventions, active social participation, and dementia education. PATIENT CONCERNS: An elderly female living at home alone in urban Beijing presented with significant short-term memory impairment, episodes of confusion, difficulty with language skills, and episodes of wandering. She had become reclusive and disengaged from her previous social networks, and no longer attended any community activities or events. The patient had no significant past medical or psychiatric history. DIAGNOSIS: The patient was diagnosed with Alzheimer's disease by a local physician based on clinical features of impaired communication, disorientation, confusion, poor judgement, behavioral changes, and difficulty speaking. Depression was considered a differential diagnosis but is also both a risk factor and symptom of dementia. INTERVENTIONS: A novel, community-based, multicomponent social care program for dementia was used to facilitate implementation of non-pharmacological interventions, gradual socialization and provide supportive carer and community education. Non-pharmacological interventions included a combination of validation therapy, music therapy, art therapy, reminiscence therapy, talking therapy, reality orientation, cognitive training, smell therapy, food therapy, sensory stimulation, garden therapy, and physiotherapy. OUTCOMES: Improvements in the patient's Geriatric Depression Scale and Mini Mental State Examination scores were noted in association with increased social participation in the community. CONCLUSION: The community-based, multicomponent dementia social care program described in this case report has enabled a socially isolated patient with Alzheimer's disease to reduce her social isolation with an associated improvement in her mood and prevention of cognitive decline. Educating the community was an essential part of re-integrating the patient into the social setting. Reducing social isolation and increasing community engagement were essential to maintaining the patient's independence in her own home.