Development and Validation of a Multiclass Model Defining Molecular Archetypes of Kidney Transplant Rejection: A Large Cohort Study of the Banff Human Organ Transplant Gene Expression Panel.

Gene expression profiling from formalin-fixed paraffin-embedded (FFPE) renal allograft biopsies is a promising approach for feasibly providing a molecular diagnosis of rejection. However, large-scale studies evaluating the performance of models using NanoString platform data to define molecular archetypes of rejection are lacking. We tested a diverse retrospective cohort of over 1400 FFPE biopsy specimens, rescored according to Banff 2019 criteria and representing 10 of 11 United Network of Organ Sharing regions, using the Banff Human Organ Transplant panel from NanoString and developed a multiclass model from the gene expression data to assign relative probabilities of 4 molecular archetypes: No Rejection, Antibody-Mediated Rejection, T Cell-Mediated Rejection, and Mixed Rejection. Using Least Absolute Shrinkage and Selection Operator regularized regression with 10-fold cross-validation fitted to 1050 biopsies in the discovery cohort and technically validated on an additional 345 biopsies, our model achieved overall accuracy of 85% in the discovery cohort and 80% in the validation cohort, with ≥75% positive predictive value for each class, except for the Mixed Rejection class in the validation cohort (positive predictive value, 53%). This study represents the technical validation of the first model built from a large and diverse sample of diagnostic FFPE biopsy specimens to define and classify molecular archetypes of histologically defined diagnoses as derived from Banff Human Organ Transplant panel gene expression profiling data.

Characteristics of complex posttraumatic stress disorder (PTSD) in young people with PTSD following multiple trauma exposure.

BACKGROUND: Complex PTSD (CPTSD) is a relatively new diagnosis. The objective of the present study was to investigate how trauma characteristics, comorbid psychopathology and cognitive and social factors experienced by children and adolescents with a posttraumatic stress disorder (PTSD) diagnosis following exposure to multiple traumatic events differs between those who meet the criteria for CPTSD and those who do not. METHOD: The present research used baseline data from the DECRYPT trial (BMJ Open, 2021, 11, e047600). Participants (n = 120) were aged 8-17 years and had exposure to multiple traumas and a PTSD diagnosis. The data collected comprised self-report and parent/caregiver-report questionnaires and interviews. Three primary analyses were conducted, comparing number of trauma types, prevalence of sexual trauma and prevalence of intrafamilial abuse between the CPTSD and PTSD-only groups. A range of comorbid psychopathology and cognitive and social factors were compared between the groups in an exploratory secondary analysis. All analyses were preregistered. RESULTS: The CPTSD group (n = 72, 60%) had a significantly higher frequency of sexual trauma than the PTSD-only group (n = 48, 40%). The groups did not significantly differ on number of trauma types or prevalence of intrafamilial abuse. From the secondary analysis, the CPTSD group were found to have significantly higher scores on measures of negative post-traumatic cognitions, depression and panic. These results were replicated in correlation analyses using a continuous measure of CPTSD symptoms. CONCLUSIONS: A large proportion of youth exposed to multiple traumatic events met criteria for CPTSD. Sexual trauma appears to be related to CPTSD symptoms. Youth with CPTSD appear to have greater severity of comorbid depression and panic symptoms, as well as more negative post-traumatic cognitions. Further investigation could focus on the directionality and mechanisms for these associations.

One session treatment (OST) is equivalent to multi-session cognitive behavioral therapy (CBT) in children with specific phobias (ASPECT): results from a national non-inferiority randomized controlled trial.

BACKGROUND: 5%-10% children and young people (CYP) experience specific phobias that impact daily functioning. Cognitive Behaviour Therapy (CBT) is recommended but has limitations. One Session Treatment (OST), a briefer alternative incorporating CBT principles, has demonstrated efficacy. The Alleviating Specific Phobias Experienced by Children Trial (ASPECT) investigated the non-inferiority of OST compared to multi-session CBT for treating specific phobias in CYP. METHODS: ASPECT was a pragmatic, multi-center, non-inferiority randomized controlled trial in 26 CAMHS sites, three voluntary agency services, and one university-based CYP well-being service. CYP aged 7-16 years with specific phobia were randomized to receive OST or CBT. Clinical non-inferiority and a nested cost-effectiveness evaluation was assessed 6-months post-randomization using the Behavioural Avoidance Task (BAT). Secondary outcome measures included the Anxiety Disorder Interview Schedule, Child Anxiety Impact Scale, Revised Children's Anxiety Depression Scale, goal-based outcome measure, and EQ-5DY and CHU-9D, collected blind at baseline and six-months. RESULTS: 268 CYPs were randomized to OST (n = 134) or CBT (n = 134). Mean BAT scores at 6 months were similar across groups in both intention-to-treat (ITT) and per-protocol (PP) populations (CBT: 7.1 (ITT, n = 76), 7.4 (PP, n = 57), OST: 7.4 (ITT, n = 73), 7.6 (PP, n = 56), on the standardized scale-adjusted mean difference for CBT compared to OST -0.123, 95% CI -0.449 to 0.202 (ITT), mean difference -0.204, 95% CI -0.579 to 0.171 (PP)). These findings were wholly below the standardized non-inferiority limit of 0.4, suggesting that OST is non-inferior to CBT. No between-group differences were found on secondary outcomes. OST marginally decreased mean service use costs and maintained similar mean Quality Adjusted Life Years compared to CBT. CONCLUSIONS: One Session Treatment has similar clinical effectiveness to CBT for specific phobias in CYP and may be a cost-saving alternative.

Clinical and cost-effectiveness of social recovery therapy for the prevention and treatment of long-term social disability among young people with emerging severe mental illness (PRODIGY): randomised controlled trial.

BACKGROUND: Young people with social disability and severe and complex mental health problems have poor outcomes, frequently struggling with treatment access and engagement. Outcomes may be improved by enhancing care and providing targeted psychological or psychosocial intervention. AIMS: We aimed to test the hypothesis that adding social recovery therapy (SRT) to enhanced standard care (ESC) would improve social recovery compared with ESC alone. METHOD: A pragmatic, assessor-masked, randomised controlled trial (PRODIGY: ISRCTN47998710) was conducted in three UK centres. Participants (n = 270) were aged 16-25 years, with persistent social disability, defined as under 30 hours of structured activity per week, social impairment for at least 6 months and severe and complex mental health problems. Participants were randomised to ESC alone or SRT plus ESC. SRT was an individual psychosocial therapy delivered over 9 months. The primary outcome was time spent in structured activity 15 months post-randomisation. RESULTS: We randomised 132 participants to SRT plus ESC and 138 to ESC alone. Mean weekly hours in structured activity at 15 months increased by 11.1 h for SRT plus ESC (mean 22.4, s.d. = 21.4) and 16.6 h for ESC alone (mean 27.7, s.d. = 26.5). There was no significant difference between arms; treatment effect was -4.44 (95% CI -10.19 to 1.31, P = 0.13). Missingness was consistently greater in the ESC alone arm. CONCLUSIONS: We found no evidence for the superiority of SRT as an adjunct to ESC. Participants in both arms made large, clinically significant improvements on all outcomes. When providing comprehensive evidence-based standard care, there are no additional gains by providing specialised SRT. Optimising standard care to ensure targeted delivery of existing interventions may further improve outcomes.

A multi-center retrospective cohort study defines the spectrum of kidney pathology in Coronavirus 2019 Disease (COVID-19).

Kidney failure is common in patients with Coronavirus Disease-19 (COVID-19), resulting in increased morbidity and mortality. In an international collaboration, 284 kidney biopsies were evaluated to improve understanding of kidney disease in COVID-19. Diagnoses were compared to five years of 63,575 native biopsies prior to the pandemic and 13,955 allograft biopsies to identify diseases that have increased in patients with COVID-19. Genotyping for APOL1 G1 and G2 alleles was performed in 107 African American and Hispanic patients. Immunohistochemistry for SARS-CoV-2 was utilized to assess direct viral infection in 273 cases along with clinical information at the time of biopsy. The leading indication for native biopsy was acute kidney injury (45.4%), followed by proteinuria with or without concurrent acute kidney injury (42.6%). There were more African American patients (44.6%) than patients of other ethnicities. The most common diagnosis in native biopsies was collapsing glomerulopathy (25.8%), which was associated with high-risk APOL1 genotypes in 91.7% of cases. Compared to the five-year biopsy database, the frequency of myoglobin cast nephropathy and proliferative glomerulonephritis with monoclonal IgG deposits was also increased in patients with COVID-19 (3.3% and 1.7%, respectively), while there was a reduced frequency of chronic conditions (including diabetes mellitus, IgA nephropathy, and arterionephrosclerosis) as the primary diagnosis. In transplants, the leading indication was acute kidney injury (86.4%), for which rejection was the predominant diagnosis (61.4%). Direct SARS-CoV-2 viral infection was not identified. Thus, our multi-center large case series identified kidney diseases that disproportionately affect patients with COVID-19 and demonstrated a high frequency of APOL1 high-risk genotypes within this group, with no evidence of direct viral infection within the kidney.

Review: Delivering mental health support within schools and colleges - a thematic synthesis of barriers and facilitators to implementation of indicated psychological interventions for adolescents.

BACKGROUND: Increasing the role of schools and colleges in the provision of mental health services for young people has the potential to improve early intervention and access to treatment. We aimed to understand what factors influence the successful implementation of indicated psychological interventions within schools and colleges to help guide increased provision of mental health support within education settings. METHODS: Systematic search for studies that have reported barriers or facilitators to the implementation of indicated interventions for adolescent emotional disorders delivered within schools and further education/sixth form colleges (CRD42018102830). Databases searched were EMBASE, MEDLINE, PsycINFO, CINAHL, British Nursing Index, ASSIA, ERIC and British Education Index. A thematic synthesis of factors reported to impact implementation was conducted. RESULTS: Two thousand five hundred and sixty-nine records and 177 full texts were screened. Fifty studies were identified for inclusion, all of which were of school-based interventions. Eleven analytic themes were developed encompassing intervention characteristics, organisational capacity, training and technical assistance, provider characteristics and community-level factors. Findings indicate the need to select appropriate interventions, consider logistical challenges of the school context and provide training and supervision to enable staff to deliver interventions with fidelity. However, structural and environmental support is required for these facilitators to have the greatest impact on successful implementation. CONCLUSIONS: Implementing indicated school-based mental health interventions is challenging. Those involved in planning school-based mental health initiatives must be alert to the impact of factors on multiple interacting levels. There is a lack of research on implementing mental health support within further education and sixth form colleges. KEY PRACTITIONER MESSAGE: Increased utilisation of schools and colleges as a setting for early intervention has been proposed as a means of improving access to mental health treatment, but successful implementation of mental health interventions within educational settings is challenging. Based on a synthesis of current evidence, we recommend that young people and education professionals should be involved in the selection of school-based interventions to ensure they are acceptable and practical to deliver within the logistical constraints of the school environment. Those delivering interventions within schools, as well as staff involved in identifying young people who might benefit from these interventions, must receive high-quality ongoing training and support. Senior school leaders play an important role in championing mental health interventions and developing a school culture that prioritises mental well-being. Health and education policy should be designed to promote a cross-sector focus on the emotional health of young people. There is a lack of evidence on the implementation of indicated psychological interventions within sixth form and further education colleges.

Detection of SARS-CoV-2 in formalin-fixed paraffin-embedded tissue sections using commercially available reagents.

Coronavirus Disease-19 (COVID-19), caused by the coronavirus SARS-CoV-2, was initially recognized in Wuhan, China and subsequently spread to all continents. The disease primarily affects the lower respiratory system, but may involve other organs and systems. Histopathologic evaluation of tissue from affected patients is crucial for diagnostic purposes, but also for advancing our understanding of the disease. For that reason, we developed immunohistochemical (IHC) and in situ hybridization (ISH) assays for detection of the. virus. A total of eight autopsy lungs, one placenta, and ten kidney biopsies from COVID-19 patients were stained with a panel of commercially available antibodies for IHC and commercially available RNA probes for ISH. Similarly, autopsy lungs, placentas and renal biopsies from non-COVID-19 patients were stained with the same antibodies and probes. All eight lungs and the placenta from COVID-19 patients stained positive by IHC and ISH, while the kidney biopsies stained negative by both methodologies. As expected, all specimens from non-COVID-19 patients were IHC and ISH negative. These two assays represent a sensitive and specific method for detecting the virus in tissue samples. We provide the protocols and the list of commercially available antibodies and probes for these assays, so they can be readily implemented in pathology laboratories and medical examiner offices for diagnostic and research purposes.

Practitioner Review: Effectiveness of indicated school-based interventions for adolescent depression and anxiety - a meta-analytic review.

BACKGROUND: Interest in delivering psychological interventions within schools to facilitate early intervention is increasing. However, most reviews have focused on universal or preventative programmes rather than interventions designed to decrease existing symptoms of depression or anxiety. This paper aims to provide a meta-analytic review of randomised controlled trials of indicated psychological interventions for young people aged 10-19 with elevated symptoms of depression and/or anxiety. METHODS: Eight electronic databases were systematically searched from inception to April 2019 for eligible trials. Study quality was assessed using two scales designed to evaluate psychotherapy intervention trials. Random effects meta-analyses were conducted separately for trials that recruited participants based on symptoms of depression and based on symptoms of anxiety. RESULTS: Data from 45 trials were analysed. Most interventions studied used cognitive and behavioural strategies. Few studies met methodological quality criteria, but effect size was not associated with study quality. Indicated school-based interventions had a small effect on reducing depression symptoms (SMD = .34, 95% CI -0.48, -0.21) and a medium effect on reducing anxiety symptoms (SMD = -.49, 95% CI -0.79, -0.19) immediately postintervention. Subgroup analyses indicated that interventions delivered by internal school staff did not have significant effects on symptoms. Reductions in depression were maintained at short-term (≤6 months) but not medium (>6 months ≤ 12) or long-term (>12-month) follow-up. Reductions in anxiety symptoms were not maintained at any follow-up. CONCLUSIONS: Indicated school-based interventions are effective at reducing symptoms of depression and anxiety in adolescents immediately postintervention but there is little evidence that these reductions are maintained. Interventions delivered by school staff are not supported by the current evidence base. Further high-quality randomised controlled trials incorporating assessment of longer-term outcomes are needed to justify increased investment in school-based interventions for adolescent depression and anxiety.

The structure of the RbBP5 ß-propeller domain reveals a surface with potential nucleic acid binding sites.

The multi-protein complex WRAD, formed by WDR5, RbBP5, Ash2L and Dpy30, binds to the MLL SET domain to stabilize the catalytically active conformation required for histone H3K4 methylation. In addition, the WRAD complex contributes to the targeting of the activated complex to specific sites on chromatin. RbBP5 is central to MLL catalytic activation, by making critical contacts with the other members of the complex. Interestingly its only major structural domain, a canonical WD40 repeat ß-propeller, is not implicated in this function. Here, we present the structure of the RbBP5 ß-propeller domain revealing a distinct, feature rich surface, dominated by clusters of Arginine residues. Our nuclear magnetic resonance binding data supports the hypothesis that in addition to the role of RbBP5 in catalytic activation, its ß-propeller domain is a platform for the recruitment of the MLL complexes to chromatin targets through its direct interaction with nucleic acids.

Evaluation of a non-diagnostic 'Psychology of Emotions' group intervention within a UK youth IAPT service: a mixed-methods approach.

BACKGROUND: A novel CBT-based intervention, tailored for young people, was developed in response to concerns about traditional diagnostically based approaches. Psychology of Emotions workshops use a normative approach to emotional difficulty instead of a diagnostic framework. AIMS: To evaluate the acceptability and efficacy of Psychology of Emotions workshops within an IAPT service for young people aged 16-25 years. METHOD: This was a mixed-methods study, evaluating routinely collected self-report measures of depression and anxiety, and qualitative feedback forms. The main outcomes were rates of attendance, change in symptom severity, and participant views of the intervention. RESULTS: From January to September 2016, 595 young people were invited to attend the Psychology of Emotions workshops, of whom 350 (58.8%) attended at least one session. Young people who attended all six sessions (8.1%) experienced significant reductions in self-reported anxiety (d = .72) and depression (d = .58) and 35.5% were classified as recovered at completion. Those who attended at least two sessions (41.3%) reported smaller but significant improvements in anxiety (d = .42) and depression (d = .45); 22.0% were classified as recovered at the last session attended. Participants provided largely positive feedback about the intervention. CONCLUSION: Psychology of Emotions is a promising treatment option, delivered outside of a diagnostic framework, for young people with mild to moderate mental health difficulties seen within IAPT services. Better understanding reasons for non-attendance might enable the intervention to be made accessible to more young people.

Safety of Algal Oil Containing EPA and DHA in cats during gestation, lactation and growth.

Algal Oil Containing EPA and DHA (AOCED) at approximately 50% was developed as a sustainable n-3 fatty acid source. AOCED was incorporated in diets at dose levels of 0%, 0.75%, 1.5% and 3.0% (w/w) and administered to healthy domestic shorthair female cats starting two weeks before mating, then during mating, gestation, lactation and to their kittens until they reached 32 weeks of age. The diets were made isocaloric and met the Association of American Feed Control Officials (AAFCO) nutrient requirements of cats for growth and reproduction. Dietary AOCED treatment did not affect the overall health, physiological parameters, food consumption and body weights of the queens and their kittens. No AOCED-related changes in haematology, coagulation or clinical chemistry parameters were observed in either generation when compared to control cats. Plasma levels of EPA and DHA were dose-dependently increased in both generations, demonstrating bioavailability of the fatty acids. In this study, safety of AOCED at levels up to 3.0% in the diet was demonstrated in cats with administration starting in utero and until kittens reached 32 weeks of age. Bioavailability of EPA and DHA in cats supports use of AOCED as a source of EPA and DHA for feline growth and reproduction.

Effect of chemotherapy on cancer stem cells and tumor-associated macrophages in a prospective study of preoperative chemotherapy in soft tissue sarcoma.

BACKGROUND: Cancer stem cells (CSC) may respond to chemotherapy differently from other tumor cells. METHODS: This study examined the expression of the putative cancer stem cell markers ALDH1, CD44, and CD133; the angiogenesis marker CD31; and the macrophage marker CD68 in soft tissue sarcomas (STS) before and after 4 cycles of chemotherapy with doxorubicin and ifosfamide in 31 patients with high-grade soft tissue sarcoma in a prospective clinical trial. RESULTS: None of the markers clearly identified CSCs in STS samples. Macrophages represented a prominent component in viable tumor areas in pre-treatment STS biopsies, ranging from < 5 to > 50%. Furthermore, macrophages expressed CD44 and ALDH1. Macrophage density correlated with baseline maximum standardized uptake value (SUVmax) on fluoro-deoxyglucose positron emission tomography (PET) imaging. Pre-chemotherapy CD68 staining correlated positively with the baseline SUVmax, and negatively with the percent of viable tumor cells in post-chemotherapy resection samples. In particular, cases with more CD68-positive cells at biopsy had fewer viable tumor cells at resection, suggesting a better response to chemotherapy. CONCLUSIONS: In conclusion, ALDH1, CD44, and CD133 are not likely to be useful markers of CSCs in STS. However, our observation of infiltrating macrophages in STS specimens indicates that these immune cells may contribute significantly to STS biology and response to chemotherapy, and could provide a potential target of therapy. Future studies should investigate macrophage contribution to STS pathophysiology by cytokine signaling.

Correlation of circular differential optical absorption with geometric chirality in plasmonic meta-atoms.

We report a strong correlation between the calculated broadband circular differential optical absorption (CDOA) and the geometric chirality of plasmonic meta-atoms with two-dimensional chirality. We investigate this correlation using three common gold meta-atom geometries: L-shapes, triangles, and nanorod dimers, over a broad range of geometric parameters. We show that this correlation holds for both contiguous plasmonic meta-atoms and non-contiguous structures which support plasmonic coupling effects. A potential application for this correlation is the rapid optimization of plasmonic nanostructure for maximum broadband CDOA.

Feasibility Using Amazon Mechanical Turk for Online Surveys of Attitudes and Perceptions of Chiropractic Health Care in the United States.

OBJECTIVE: The purpose of this study was to determine the feasibility and utility of using Amazon Mechanical Turk (MTurk) for online surveys of US adults about their perceptions of chiropractors and to report differences between people who have positive versus negative attitudes toward chiropractic care. METHODS: A 74-item, unvalidated survey was developed through iterative review to assess attitudes of respondents to chiropractic, including a query stratifying respondents based on previous and future use of chiropractic (have used, would consider; have used, would not consider; have not used, would consider; have not used, would not consider). The electronic survey was delivered using Qualtrics; respondents were recruited using MTurk, a crowdsourcing website. Descriptive statistics, including frequencies and cross tabulations, were performed. RESULTS: A total of 1300 responses were obtained. Consistent with previous reports, 32.2% of the respondents reported having seen a chiropractor in the past. Chiropractic care was perceived as being effective for musculoskeletal complaints. Respondents who would not consider future chiropractic care shared a common set of beliefs related to training of chiropractors, scope of chiropractic practice, and safety and reputation of chiropractic. These respondents reported increased likelihood of chiropractor use with the recommendation of a primary care physician. CONCLUSION: Recruiting survey participants using MTurk is feasible, affordable, and quick and offers high utility to academic researchers. Using this resource, we ascertained preliminary data about attitudes and perceptions from individuals who would or would not consider chiropractic, stratified by their previous use.

Genetic testing of complement and coagulation pathways in patients with severe hypertension and renal microangiopathy.

A diagnosis of thrombotic microangiopathy on kidney biopsy in a patient presenting with hypertensive emergency has historically elicited the diagnosis of malignant hypertension-associated thrombotic microangiopathy. Recent studies, however, have raised awareness that a number of these patients may actually represent atypical hemolytic uremic syndrome. To further investigate this premise, we performed next-generation sequencing to interrogate the coding regions of 29 complement and coagulation cascade genes associated with atypical hemolytic uremic syndrome in 100 non-elderly patients presenting with severe hypertension, renal failure and a kidney biopsy showing microangiopathic changes limited to the classic accelerated hypertension-associated lesion of arterial intimal edema ('mucoid intimal hyperplasia') in isolation and without accompanying glomerular microthrombi. No pathogenic or likely pathogenic variants were identified in any of the genes analyzed, although 13 patients had rare variants of uncertain significance predicted to be deleterious by all in-silico prediction methods utilized. Accordingly, this large patient cohort showed no definitive burden of disease secondary to genetic variants involving complement or coagulation pathways, which contrasts sharply with the high frequency of similar mutational events reported for atypical hemolytic uremic syndrome. Our results also inform recent data by suggesting that patients who present with severe or malignant hypertension and renal thrombotic microangiopathy may be at higher risk for atypical hemolytic uremic syndrome only if the biopsy shows more active disease that includes glomerular fibrin thrombi.

Second-generation antipsychotic long-acting injections in bipolar disorder: Systematic review and meta-analysis.

BACKGROUND: Non-adherence is a significant problem in bipolar disorder. Second-generation antipsychotics (SGA) long-acting injections (LAIs) may improve adherence in bipolar disorder and may prevent relapses. However, the evidence is limited and conflicting. OBJECTIVE: The objective of this study was to evaluate efficacy and safety of SGA LAIs in bipolar disorder. METHOD: Systematic review and meta-analysis of randomised controlled trials (RCTs) (≥6 months duration) investigating safety and efficacy of SGA LAIs for bipolar disorder. We searched Pubmed, Embase, CINAHL, Cochrane, PsycINFO, LiLACS, www.clinicaltrials.gov up to October 2016. We also contacted the manufacturers of SGA LAIs. Primary efficacy and safety outcomes were relapse rate and all-cause discontinuation respectively. RESULTS: Total of seven RCTs (n = 1192) were included. SGA LAIs show superiority over placebo for study-defined relapse rate (RR = 0.58, 95% CI = 0.49-0.68, P < 0.00001) and all-cause discontinuation (RR = 0.72, 95% CI = 0.64-0.82, P < 0.00001). However, no significant difference was found between SGA LAIs and oral active control for relapse rate (RR = 0.92, P = 0.79) and all-cause discontinuation (RR = 1.2, P = 0.31). In terms of secondary outcomes, SGA LAIs performed better than placebo in relapse to mania/hypomania, young mania rating scales (YMRS), clinical global impression-severity (CGI-S), montgomery-asberg depression rating scale (MADRS). There was no significant difference between SGA LAIs and oral active control regarding relapse to mania/hypomania, YMRS, CGI-S, extra-pyramidal side effects (EPSEs), weight gain. However, the active control performed better than SGA LAIs in relapse to depression, MADRS, and prolactin-related AEs. CONCLUSIONS: Current evidence is very limited to support the use of SGA LAIs (compared to oral medication) in bipolar disorder. Further high-quality studies, particularly comparing SGA LAIs with active control, are warranted.

Phosphorylation of AMPK by upstream kinases is required for activity in mammalian cells.

AMP-activated protein kinase (AMPK) plays a major role in regulating metabolism and has attracted significant attention as a therapeutic target for treating metabolic disorders. AMPK activity is stimulated more than 100-fold by phosphorylation of threonine 172 (Thr172). Binding of AMP to the γ subunit allosterically activates the kinase. Additionally, many small molecules, e.g. 991, have been identified that bind between the kinase domain and the carbohydrate-binding module of the ß subunit, stabilising their interaction and leading to activation. It was reported recently that non-phosphorylated Thr172 AMPK is activated by AMP and A769662. We present here the crystal structure of non-phosphorylated Thr172 AMPK in complex with AMP and 991. This structure reveals that the activation loop, as well as the complex overall, is similar to the Thr172 phosphorylated complex. We find that in the presence of AMP and 991 non-phosphorylated Thr172, AMPK is much less active than the Thr172 phosphorylated enzyme. In human cells, the basal level of Thr172 phosphorylation is very low (∼1%), but is increased 10-fold by treatment with 2-deoxyglucose. In cells lacking the major Thr172 kinases, LKB1 and CaMKKß, Thr172 phosphorylation is almost completely abolished, and AMPK activity is virtually undetectable. Our data show that AMP and 991 binding to non-phosphorylated Thr172 AMPK can induce an ordered, active-like, conformation of the activation loop explaining how AMPK activity can be measured in vitro without Thr172 phosphorylation. However, in a cellular context, phosphorylation of Thr172 is critical for significant activation of AMPK.

Clinician-rated and self-reported psychotic-like experiences in individuals accessing a specialist Youth Mental Health Service.

AIM: The prevalence of psychotic-like experiences (PLEs) was explored in a sample of 14- to 25-year-olds with non-psychotic mental health difficulties. Associations between PLEs, psychopathology, functioning, trauma history, and pathways to care were examined. METHODS: Data were collected for 202 young people. Clinicians rated PLEs using the Primary Care Checklist (PCC) and functioning using Global Assessment Scales. Eighty-three young people completed self-report assessments of PLEs using the Prodromal Questionnaire (PQ-16) and measures of social anxiety, depression, trauma history, and pathways to care. RESULTS: There was a high prevalence of PLEs in the sample. The prevalence of PLEs was higher when young people self-rated their experiences. Endorsement frequencies for PLEs ranged from 3.5 to 24% on the PCC and 22 to 70% on the PQ-16. Higher scores on the PQ-16 were associated with more pathways into care and greater exposure to traumatic life events. CONCLUSIONS: PLEs are common in young people with non-psychotic mental health difficulties and may reflect increased severity and complexity of mental health difficulties. Routine screening and further assessment of PLEs are important in understanding and responding to such experiences. Screening should include self-rating of PLEs as well as clinician-rated scales. PRACTITIONER POINTS: Clinical Implications Psychotic-like experiences are common in young people with severe non-psychotic mental health problems and should be routinely screened in mental health services. Psychotic-like experiences were found to be more prevalent when using a self-report screening tool compared to a clinician-rated measure. The presence of psychotic-like experiences may reflect more severe and complex mental health problems and may also cause delays in young people accessing the right kind of support. ;Limitations This study only assessed the presence or absence of psychotic-like experiences. Further studies should use more detailed assessments to understand more about the nature of such experiences and how they are appraised and responded to. This study is cross-sectional, and therefore, the direction of the relationships between psychotic-like experiences and depression, anxiety, functioning, and trauma cannot be specified. Longitudinal studies are needed to examine the impact of psychotic-like experiences on long-term outcomes.

Structural basis for the requirement of additional factors for MLL1 SET domain activity and recognition of epigenetic marks.

The mixed-lineage leukemia protein MLL1 is a transcriptional regulator with an essential role in early development and hematopoiesis. The biological function of MLL1 is mediated by the histone H3K4 methyltransferase activity of the carboxyl-terminal SET domain. We have determined the crystal structure of the MLL1 SET domain in complex with cofactor product AdoHcy and a histone H3 peptide. This structure indicates that, in order to form a well-ordered active site, a highly variable but essential component of the SET domain must be repositioned. To test this idea, we compared the effect of the addition of MLL complex members on methyltransferase activity and show that both RbBP5 and Ash2L but not Wdr5 stimulate activity. Additionally, we have determined the effect of posttranslational modifications on histone H3 residues downstream and upstream from the target lysine and provide a structural explanation for why H3T3 phosphorylation and H3K9 acetylation regulate activity.