Exploration of the P1 residue in 3CL protease inhibitors leading to the discovery of a 2-tetrahydrofuran P1 replacement.

The virally encoded 3C-like protease (3CLpro) is a well-validated drug target for the inhibition of coronaviruses including Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). Most inhibitors of 3CLpro are peptidomimetic, with a γ-lactam in place of Gln at the P1 position of the pseudopeptide chain. An effort was pursued to identify a viable alternative to the γ-lactam P1 mimetic which would improve physicochemical properties while retaining affinity for the target. Discovery of a 2-tetrahydrofuran as a suitable P1 replacement that is a potent enzymatic inhibitor of 3CLpro in SARS-CoV-2 virus is described herein.

Suppression of the gene encoding PDZ domain-containing protein decreases cold tolerance and overwintering survival of the mosquito, Culex pipiens (Culicidae: Diptera).

In diapausing mosquitoes, cold tolerance and prolonged lifespan are important features that are crucial for overwintering success. In the mosquito Culex pipiens, we suggest that PDZ domain-containing protein (PDZ) (post synaptic density protein [PSD95], drosophila disc large tumor suppressor [Dlg1], and zonula occludens-1 protein [zo-1]) domain-containing protein is involved with these diapause features for overwintering survival in Culex mosquitoes. The expression level of pdz was significantly higher in diapausing adult females in the early stage in comparison to their nondiapausing counterparts. Suppression of the gene that encodes PDZ by RNA interference significantly decreased actin accumulation in the midgut of early-stage adult diapausing females. Inhibition of pdz also significantly reduced the survivability of diapausing females which indicates that this protein could play a key role in preserving the midgut tissues during early diapause.

Autism Spectrum Disorder: A Review.

Importance: Autism spectrum disorder (ASD), characterized by deficits in social communication and the presence of restricted, repetitive behaviors or interests, is a neurodevelopmental disorder affecting approximately 2.3% children aged 8 years in the US and approximately 2.2% of adults. This review summarizes evidence on the diagnosis and treatment of ASD. Observations: The estimated prevalence of ASD has been increasing in the US, from 1.1% in 2008 to 2.3% in 2018, which is likely associated with changes in diagnostic criteria, improved performance of screening and diagnostic tools, and increased public awareness. No biomarkers specific to the diagnosis of ASD have been identified. Common early signs and symptoms of ASD in a child's first 2 years of life include no response to name when called, no or limited use of gestures in communication, and lack of imaginative play. The criterion standard for the diagnosis of ASD is a comprehensive evaluation with a multidisciplinary team of clinicians and is based on semistructured direct observation of the child's behavior and semistructured caregiver interview focused on the individual's development and behaviors using standardized measures, such as the Autism Diagnostic Observation Schedule-Second Edition and the Autism Diagnostic Interview. These diagnostic measures have sensitivity of 91% and 80% and specificity of 76% and 72%, respectively. Compared with people without ASD, individuals with ASD have higher rates of depression (20% vs 7%), anxiety (11% vs 5%), sleep difficulties (13% vs 5%), and epilepsy (21% with co-occurring intellectual disability vs 0.8%). Intensive behavioral interventions, such as the Early Start Denver Model, are beneficial in children 5 years or younger for improvement in language, play, and social communication (small to medium effect size based on standardized mean difference). Pharmacotherapy is indicated for co-occurring psychiatric conditions, such as emotion dysregulation or attention-deficit/hyperactivity disorder. Risperidone and aripiprazole can improve irritability and aggression (standardized mean difference of 1.1, consistent with a large effect size) compared with placebo. Psychostimulants are effective for attention-deficit/hyperactivity disorder (standardized mean difference of 0.6, consistent with a moderate effect size) compared with placebo. These medications are associated with adverse effects including, most commonly, changes in appetite, weight, and sleep. Conclusions and Relevance: ASD affects approximately 2.3% of children aged 8 years and approximately 2.2% of adults in the US. First-line therapy consists of behavioral interventions, while co-occurring psychiatric conditions, such as anxiety or aggression, may be treated with specific behavioral therapy or medication.

Genetic and epigenetic signatures associated with plasma oxytocin levels in children and adolescents with autism spectrum disorder.

Oxytocin (OT), the brain's most abundant neuropeptide, plays an important role in social salience and motivation. Clinical trials of the efficacy of OT in autism spectrum disorder (ASD) have reported mixed results due in part to ASD's complex etiology. We investigated whether genetic and epigenetic variation contribute to variable endogenous OT levels that modulate sensitivity to OT therapy. To carry out this analysis, we integrated genome-wide profiles of DNA-methylation, transcriptional activity, and genetic variation with plasma OT levels in 290 participants with ASD enrolled in a randomized controlled trial of OT. Our analysis identified genetic variants with novel association with plasma OT, several of which reside in known ASD risk genes. We also show subtle but statistically significant association of plasma OT levels with peripheral transcriptional activity and DNA-methylation profiles across several annotated gene sets. These findings broaden our understanding of the effects of the peripheral oxytocin system and provide novel genetic candidates for future studies to decode the complex etiology of ASD and its interaction with OT signaling and OT-based interventions. LAY SUMMARY: Oxytocin (OT) is an abundant chemical produced by neurons that plays an important role in social interaction and motivation. We investigated whether genetic and epigenetic factors contribute to variable OT levels in the blood. To this, we integrated genetic, gene expression, and non-DNA regulated (epigenetic) signatures with blood OT levels in 290 participants with autism enrolled in an OT clinical trial. We identified genetic association with plasma OT, several of which reside in known autism risk genes. We also show statistically significant association of plasma OT levels with gene expression and epigenetic across several gene pathways. These findings broaden our understanding of the factors that influence OT levels in the blood for future studies to decode the complex presentation of autism and its interaction with OT and OT-based treatment.

Adaptive Behavior in Young Autistic Children: Associations with Irritability and ADHD Symptoms.

Attention-deficit/hyperactivity disorder (ADHD) symptoms affect 40-60% of autistic children and have been linked to differences in adaptive behavior. It is unclear whether adaptive behavior in autistic youth is directly impacted by co-occurring ADHD symptoms or by another associated feature of both autism and ADHD, such as increased irritability. The current study examined relationships between irritability, ADHD symptoms, and adaptive behavior in 3- to 7-year-old autistic children. Results suggest that, after adjusting for co-occurring ADHD symptoms, higher levels of irritability are associated with differences in social adaptive behavior specifically. Understanding relationships between irritability, ADHD, and adaptive behavior in autistic children is critical because measures of adaptive behavior, such as the Vineland Scales of Adaptive Functioning, are often used as a proxy for global functioning, as well as for developing intervention plans and measuring outcomes as primary endpoints in clinical trials.

Adolescents With Eating Disorders in Pediatric Practice - The European Academy of Paediatrics Recommendations.

In the face of the growing number of adolescents suffering from eating disorders (EDs) and access to psychiatric care limited by the epidemiological and demographic situation, the primary care pediatrician's role in diagnosing and treating EDs is growing. The European Academy of Paediatrics (EAP) decided to summarize knowledge about EDs and formulate recommendations to support European pediatricians and improve care for adolescents with EDs.

Citalopram Did Not Significantly Improve Anxiety in Children with Autism Spectrum Disorder Undergoing Treatment for Core Symptoms: Secondary Analysis of a Trial to Reduce Repetitive Behaviors.

Objective: Anxiety disorders are among the most common co-occurring conditions in autism spectrum disorder (ASD). Despite their prevalence and impact, there are no randomized controlled trials (RCTs) aimed at evaluating the efficacy of selective serotonin reuptake inhibitors (SSRIs) for anxiolysis in this population, who may have a different biological basis for anxiety. Methods: Secondary analyses of the STAART double-blind, placebo-controlled RCT of citalopram in children with ASD examined whether citalopram reduced anxiety measured on the parent-reported Child and Adolescent Symptom Inventory-4 (CASI-4) as the primary outcome. An intention-to-treat analysis involving all 149 participants used multiple imputations for missing data and included baseline stratification factors of age group and site, among others. We prespecified as clinically significant a 33% reduction in anxiety in citalopram versus placebo, coinciding with 80% power. We tested whether communicative ability on the Vineland Communication score moderated treatment effect and explored whether initial anxiety was associated with greater adverse events, which could impact on dose titration and achieving optimal dose. Results: Both groups showed substantial reduction in anxiety. Citalopram was associated with a nonsignificant 16.5% greater reduction (observed coefficient = -0.181, bootstrap standard error = 0.126, p = 0.151, confidence interval = -0.428 to 0.066). Anxiety reports were significantly lower in children with reduced communicative ability, but communicative ability did not moderate the treatment effect (interaction p = 0.294). Initial anxiety levels were not associated with increased adverse effects (interaction ps 0.162-0.954). Conclusion: Citalopram did not statistically significantly improve anxiety in children with ASD. Clinicians should be cautious in their use of SSRIs for this indication. There remains a need for well-powered clinical trials testing the efficacy of SSRIs among autistic children with anxiety disorders.

Structural characterization of dicyanopyridine containing DNMT1-selective, non-nucleoside inhibitors.

DNMT1 maintains the parental DNA methylation pattern on newly replicated hemimethylated DNA. The failure of this maintenance process causes aberrant DNA methylation that affects transcription and contributes to the development and progression of cancers such as acute myeloid leukemia. Here, we structurally characterized a set of newly discovered DNMT1-selective, reversible, non-nucleoside inhibitors that bear a core 3,5-dicyanopyridine moiety, as exemplified by GSK3735967, to better understand their mechanism of inhibition. All of the dicyanopydridine-containing inhibitors examined intercalate into the hemimethylated DNA between two CpG base pairs through the DNA minor groove, resulting in conformational movement of the DNMT1 active-site loop. In addition, GSK3735967 introduces two new binding sites, where it interacts with and stabilizes the displaced DNMT1 active-site loop and it occupies an open aromatic cage in which trimethylated histone H4 lysine 20 is expected to bind. Our work represents a substantial step in generating potent, selective, and non-nucleoside inhibitors of DNMT1.

Fibroblast pyruvate carboxylase is required for collagen production in the tumour microenvironment.

The aberrant production of collagen by fibroblasts is a hallmark of many solid tumours and can influence cancer progression. How the mesenchymal cells in the tumour microenvironment maintain their production of extracellular matrix proteins as the vascular delivery of glutamine and glucose becomes compromised remains unclear. Here we show that pyruvate carboxylase (PC)-mediated anaplerosis in tumour-associated fibroblasts contributes to tumour fibrosis and growth. Using cultured mesenchymal and cancer cells, as well as mouse allograft models, we provide evidence that extracellular lactate can be utilized by fibroblasts to maintain tricarboxylic acid (TCA) cycle anaplerosis and non-essential amino acid biosynthesis through PC activity. Furthermore, we show that fibroblast PC is required for collagen production in the tumour microenvironment. These results establish TCA cycle anaplerosis as a determinant of extracellular matrix collagen production, and identify PC as a potential target to inhibit tumour desmoplasia.

Intranasal Oxytocin in Children and Adolescents with Autism Spectrum Disorder.

BACKGROUND: Experimental studies and small clinical trials have suggested that treatment with intranasal oxytocin may reduce social impairment in persons with autism spectrum disorder. Oxytocin has been administered in clinical practice to many children with autism spectrum disorder. METHODS: We conducted a 24-week, placebo-controlled phase 2 trial of intranasal oxytocin therapy in children and adolescents 3 to 17 years of age with autism spectrum disorder. Participants were randomly assigned in a 1:1 ratio, with stratification according to age and verbal fluency, to receive oxytocin or placebo, administered intranasally, with a total target dose of 48 international units daily. The primary outcome was the least-squares mean change from baseline on the Aberrant Behavior Checklist modified Social Withdrawal subscale (ABC-mSW), which includes 13 items (scores range from 0 to 39, with higher scores indicating less social interaction). Secondary outcomes included two additional measures of social function and an abbreviated measure of IQ. RESULTS: Of the 355 children and adolescents who underwent screening, 290 were enrolled. A total of 146 participants were assigned to the oxytocin group and 144 to the placebo group; 139 and 138 participants, respectively, completed both the baseline and at least one postbaseline ABC-mSW assessments and were included in the modified intention-to-treat analyses. The least-squares mean change from baseline in the ABC-mSW score (primary outcome) was -3.7 in the oxytocin group and -3.5 in the placebo group (least-squares mean difference, -0.2; 95% confidence interval, -1.5 to 1.0; P = 0.61). Secondary outcomes generally did not differ between the trial groups. The incidence and severity of adverse events were similar in the two groups. CONCLUSIONS: This placebo-controlled trial of intranasal oxytocin therapy in children and adolescents with autism spectrum disorder showed no significant between-group differences in the least-squares mean change from baseline on measures of social or cognitive functioning over a period of 24 weeks. (Funded by the National Institute of Child Health and Human Development; SOARS-B ClinicalTrials.gov number, NCT01944046.).

Inducible and reversible inhibition of miRNA-mediated gene repression in vivo.

Although virtually all gene networks are predicted to be controlled by miRNAs, the contribution of this important layer of gene regulation to tissue homeostasis in adult animals remains unclear. Gain and loss-of-function experiments have provided key insights into the specific function of individual miRNAs, but effective genetic tools to study the functional consequences of global inhibition of miRNA activity in vivo are lacking. Here we report the generation and characterization of a genetically engineered mouse strain in which miRNA-mediated gene repression can be reversibly inhibited without affecting miRNA biogenesis or abundance. We demonstrate the usefulness of this strategy by investigating the consequences of acute inhibition of miRNA function in adult animals. We find that different tissues and organs respond differently to global loss of miRNA function. While miRNA-mediated gene repression is essential for the homeostasis of the heart and the skeletal muscle, it is largely dispensable in the majority of other organs. Even in tissues where it is not required for homeostasis, such as the intestine and hematopoietic system, miRNA activity can become essential during regeneration following acute injury. These data support a model where many metazoan tissues primarily rely on miRNA function to respond to potentially pathogenic events.

DNA-Encoded Library Hit Confirmation: Bridging the Gap Between On-DNA and Off-DNA Chemistry.

DNA-encoded library (DEL) technology is a powerful platform for hit identification in academia and the pharmaceutical industry. When conducting off-DNA resynthesis hit confirmation after affinity selection, PCR/sequencing, and data analysis, one typically assumes a "one-to-one" relationship between the DNA tag and the chemical structure of the attached small-molecule it encodes. Because library synthesis often yields a mixture, this approximation increases the risk of overlooking positive discoveries and valuable information. To address this issue, we apply a library synthesis "recipe" strategy for on-DNA resynthesis using a cleavable linker, followed by direct affinity selection mass spectrometry (AS-MS) evaluation and identification of binder(s) from the released small-molecule mixture. We validate and showcase this approach employing the receptor-interacting-protein kinase 2 (RIP2) DEL campaign. We also designed and developed two cleavable linkers to enable this method, a photocleavable linker (nitrophenyl-based) and acid-labile linker (tetrahydropyranyl ether). The strategy provides an effective means of hit identification and rapid determination of key active component(s) of the mixture.

Suppressed expression of oxidoreductin-like protein, Oxidor, increases follicle degeneration and decreases survival during the overwintering diapause of the mosquito Culex pipiens.

Throughout diapause in mosquitoes, stress resistance and subsequent prolonged lifespan are a few important features of diapause that are crucial for overwintering success. In the mosquito Culex pipiens, we suggest that oxidoreductin-like protein is involved with these diapause characteristics for overwintering survival. Expression of oxidor was more than two-fold higher in early stage diapausing females compared to their non-diapausing counterparts. Suppression of the gene that encodes oxidoreductin-like protein by RNAi significantly increased the proportion of degenerating follicles in early-stage adult diapausing females. Inhibition of oxidor also significantly reduced the survivability of diapausing females which indicates that this protein plays a key role in protecting multiple tissues during early diapause.

Predictors of Caregiver Strain for Parents of Children with Autism Spectrum Disorder.

Parents of children with autism spectrum disorder (ASD) face higher levels of caregiver strain compared to parents of children with other disabilities. This study examined child clinical features that predict high levels of caregiver strain for 374 parents of children with ASD. Caregiver strain was measured using the Caregiver Strain Questionnaire (CGSQ) objective, subjective internalized, and subjective externalized subscales. Confirmatory factor analysis indicated an acceptable fit for the original CGSQ three-factor solution. The strongest child predictors across CGSQ subscales were: disruptive behavior for objective strain, autism severity and disruptive behavior for subjective internalized strain, and oppositional behavior and hyperactivity for subjective externalized strain. Individualized interventions that attend to specific elements of parental strain may reduce strain and improve family wellbeing.

Translation in amino-acid-poor environments is limited by tRNAGln charging.

An inadequate supply of amino acids leads to accumulation of uncharged tRNAs, which can bind and activate GCN2 kinase to reduce translation. Here, we show that glutamine-specific tRNAs selectively become uncharged when extracellular amino acid availability is compromised. In contrast, all other tRNAs retain charging of their cognate amino acids in a manner that is dependent upon intact lysosomal function. In addition to GCN2 activation and reduced total translation, the reduced charging of tRNAGln in amino-acid-deprived cells also leads to specific depletion of proteins containing polyglutamine tracts including core-binding factor α1, mediator subunit 12, transcriptional coactivator CBP and TATA-box binding protein. Treating amino-acid-deprived cells with exogenous glutamine or glutaminase inhibitors restores tRNAGln charging and the levels of polyglutamine-containing proteins. Together, these results demonstrate that the activation of GCN2 and the translation of polyglutamine-encoding transcripts serve as key sensors of glutamine availability in mammalian cells.

Yap/Taz promote the scavenging of extracellular nutrients through macropinocytosis.

The uptake of macromolecules and cellular debris through macropinocytosis has emerged as an important nutrient acquisition strategy of cancer cells. Genetic alterations commonly found in human cancers (e.g. mutations in KRAS or loss of PTEN) have been shown to increase macropinocytosis. To identify additional effectors that enable cell growth dependent on the uptake of extracellular proteins, pancreatic ductal adenocarcinoma (PDA) cells were selected for growth in medium where extracellular albumin was the obligate source of the essential amino acid leucine. Analysis of global changes in chromatin availability and gene expression revealed that PDA cells selected under these conditions exhibited elevated activity of the transcriptional activators Yap/Taz. Knockout of Yap/Taz prevented growth of PDA cells in leucine-deficient medium, but not in complete medium. Furthermore, constitutively active forms of Yap or Taz were sufficient to stimulate macropinocytosis of extracellular protein. In addition to promoting the uptake of plasma proteins, Yap/Taz also promoted the scavenging of apoptotic cell bodies and necrotic debris by PDA cells. The Yap/Taz transcriptional target Axl was found to be essential for cell growth dependent on the uptake of dead cells and cell debris. Together, these studies suggest that the Hippo pathway effectors Yap and Taz are important transcriptional regulators of endocytic nutrient uptake.

Rationale, design, and methods of the Autism Centers of Excellence (ACE) network Study of Oxytocin in Autism to improve Reciprocal Social Behaviors (SOARS-B).

OBJECTIVE: To describe the rationale, design, and methods of the Autism Centers of Excellence (ACE) network Study of Oxytocin in Autism to improve Reciprocal Social Behaviors (SOARS-B). METHOD: This phase 2 clinical trial was designed to evaluate the use of intranasal oxytocin treatment to improve social difficulties in individuals with autism spectrum disorder (ASD). In total, 290 participants ages 3 to 17 years with a DSM-5 diagnosis of ASD were enrolled to receive 24 weeks of treatment with either oxytocin or a matched placebo at one of seven collaborating sites. Participants were subsequently treated with open-label oxytocin for 24 additional weeks. Post-treatment assessments were done approximately 4 weeks after treatment discontinuation. Plasma oxytocin and oxytocin receptor gene (OXTR) methylation level were measured at baseline, and week 8, 24 and 36 to explore potential relationships between these biomarkers and treatment response. RESULTS: This report describes the rationale, design, and methods of the SOARS-B clinical trial. CONCLUSIONS: There is a tremendous unmet need for safe and effective pharmacological treatment options that target the core symptoms of ASD. Several studies support the hypothesis that intranasal oxytocin could improve social orienting and the salience of social rewards in ASD, thereby enhancing reciprocal social behaviors. However, due to conflicting results from a number of pilot studies on the prosocial effects of exogenous oxytocin, this hypothesis remains controversial and inconclusive. SOARS-B is the best powered study to date to address this hypothesis and promises to improve our understanding of the safety and efficacy of intranasal oxytocin in the treatment of social deficits in children with ASD.

Impaired mitochondrial oxidative phosphorylation limits the self-renewal of T cells exposed to persistent antigen.

The majority of tumor-infiltrating T cells exhibit a terminally exhausted phenotype, marked by a loss of self-renewal capacity. How repetitive antigenic stimulation impairs T cell self-renewal remains poorly defined. Here, we show that persistent antigenic stimulation impaired ADP-coupled oxidative phosphorylation. The resultant bioenergetic compromise blocked proliferation by limiting nucleotide triphosphate synthesis. Inhibition of mitochondrial oxidative phosphorylation in activated T cells was sufficient to suppress proliferation and upregulate genes linked to T cell exhaustion. Conversely, prevention of mitochondrial oxidative stress during chronic T cell stimulation allowed sustained T cell proliferation and induced genes associated with stem-like progenitor T cells. As a result, antioxidant treatment enhanced the anti-tumor efficacy of chronically stimulated T cells. These data reveal that loss of ATP production through oxidative phosphorylation limits T cell proliferation and effector function during chronic antigenic stimulation. Furthermore, treatments that maintain redox balance promote T cell self-renewal and enhance anti-tumor immunity.

High-Resolution In Vivo Identification of miRNA Targets by Halo-Enhanced Ago2 Pull-Down.

The identification of microRNA (miRNA) targets by Ago2 crosslinking-immunoprecipitation (CLIP) methods has provided major insights into the biology of this important class of non-coding RNAs. However, these methods are technically challenging and not easily applicable to an in vivo setting. To overcome these limitations and facilitate the investigation of miRNA functions in vivo, we have developed a method based on a genetically engineered mouse harboring a conditional Halo-Ago2 allele expressed from the endogenous Ago2 locus. By using a resin conjugated to the HaloTag ligand, Ago2-miRNA-mRNA complexes can be purified from cells and tissues expressing the endogenous Halo-Ago2 allele. We demonstrate the reproducibility and sensitivity of this method in mouse embryonic stem cells, developing embryos, adult tissues, and autochthonous mouse models of human brain and lung cancers. This method and the datasets we have generated will facilitate the characterization of miRNA-mRNA networks in vivo under physiological and pathological conditions.