Vaccination induces broadly neutralizing antibody precursors to HIV gp41.

A key barrier to the development of vaccines that induce broadly neutralizing antibodies (bnAbs) against human immunodeficiency virus (HIV) and other viruses of high antigenic diversity is the design of priming immunogens that induce rare bnAb-precursor B cells. The high neutralization breadth of the HIV bnAb 10E8 makes elicitation of 10E8-class bnAbs desirable; however, the recessed epitope within gp41 makes envelope trimers poor priming immunogens and requires that 10E8-class bnAbs possess a long heavy chain complementarity determining region 3 (HCDR3) with a specific binding motif. We developed germline-targeting epitope scaffolds with affinity for 10E8-class precursors and engineered nanoparticles for multivalent display. Scaffolds exhibited epitope structural mimicry and bound bnAb-precursor human naive B cells in ex vivo screens, protein nanoparticles induced bnAb-precursor responses in stringent mouse models and rhesus macaques, and mRNA-encoded nanoparticles triggered similar responses in mice. Thus, germline-targeting epitope scaffold nanoparticles can elicit rare bnAb-precursor B cells with predefined binding specificities and HCDR3 features.

Passive Transfer of Vaccine-Elicited Antibodies Protects against SIV in Rhesus Macaques.

Several HIV-1 and SIV vaccine candidates have shown partial protection against viral challenges in rhesus macaques. However, the protective efficacy of vaccine-elicited polyclonal antibodies has not previously been demonstrated in adoptive transfer studies in nonhuman primates. In this study, we show that passive transfer of purified antibodies from vaccinated macaques can protect naive animals against SIVmac251 challenges. We vaccinated 30 rhesus macaques with Ad26-SIV Env/Gag/Pol and SIV Env gp140 protein vaccines and assessed the induction of antibody responses and a putative protective signature. This signature included multiple antibody functions and correlated with upregulation of interferon pathways in vaccinated animals. Adoptive transfer of purified immunoglobulin G (IgG) from the vaccinated animals with the most robust protective signatures provided partial protection against SIVmac251 challenges in naive recipient rhesus macaques. These data demonstrate the protective efficacy of purified vaccine-elicited antiviral antibodies in this model, even in the absence of virus neutralization.

Chemi-Northern: a versatile chemiluminescent northern blot method for analysis and quantitation of RNA molecules.

This report describes a chemiluminescence-based detection method for RNAs on northern blots, designated Chemi-Northern. This approach builds on the simplicity and versatility of northern blotting, while dispensing of the need for expensive and cumbersome radioactivity. RNAs are first separated by denaturing gel electrophoresis, transferred to a nylon membrane, and then hybridized to a biotinylated RNA or DNA antisense probe. Streptavidin conjugated with horseradish peroxidase and enhanced chemiluminescence substrate are then used to detect the probe bound to the target RNA. Our results demonstrate the versatility of this method in detecting natural and engineered RNAs expressed in cells, including messenger and noncoding RNAs. We show that Chemi-Northern detection is sensitive and fast, detecting attomole amounts of RNA in as little as 1 sec, with high signal intensity and low background. The dynamic response displays excellent linearity. Using Chemi-Northern, we measure the reproducible, statistically significant reduction of mRNA levels by human sequence-specific RNA-binding proteins, PUM1 and PUM2. Additionally, we measure the interaction of the poly(A) binding protein, PABPC1, with polyadenylated mRNA. Thus, the Chemi-Northern method provides a versatile, simple, and cost-effective method to enable researchers to analyze expression, processing, binding, and decay of RNAs.

Editing and methylation at a single site by functionally interdependent activities.

Nucleic acids undergo naturally occurring chemical modifications. Over 100 different modifications have been described and every position in the purine and pyrimidine bases can be modified; often the sugar is also modified. Despite recent progress, the mechanism for the biosynthesis of most modifications is not fully understood, owing, in part, to the difficulty associated with reconstituting enzyme activity in vitro. Whereas some modifications can be efficiently formed with purified components, others may require more intricate pathways. A model for modification interdependence, in which one modification is a prerequisite for another, potentially explains a major hindrance in reconstituting enzymatic activity in vitro. This model was prompted by the earlier discovery of tRNA cytosine-to-uridine editing in eukaryotes, a reaction that has not been recapitulated in vitro and the mechanism of which remains unknown. Here we show that cytosine 32 in the anticodon loop of Trypanosoma brucei tRNAThr is methylated to 3-methylcytosine (m3C) as a pre-requisite for C-to-U deamination. Formation of m3C in vitro requires the presence of both the T. brucei m3C methyltransferase TRM140 and the deaminase ADAT2/3. Once formed, m3C is deaminated to 3-methyluridine (m3U) by the same set of enzymes. ADAT2/3 is a highly mutagenic enzyme, but we also show that when co-expressed with the methyltransferase its mutagenicity is kept in check. This helps to explain how T. brucei escapes 'wholesale deamination' of its genome while harbouring both enzymes in the nucleus. This observation has implications for the control of another mutagenic deaminase, human AID, and provides a rationale for its regulation.

Parent Management Training Augmentation to Address Coercive and Disruptive Behavior in Cognitive-Behavioral Therapy for Pediatric Obsessive-Compulsive Disorder.

Coercive and disruptive behaviors commonly interfere with cognitive-behavioral therapy (CBT) trials among youths with obsessive-compulsive disorder (OCD). Although evidence supports parent management training (PMT) for reducing disruptive behavior, no group-based PMT interventions exist for OCD-related disruptive behaviors. We studied feasibility and effectiveness of group-based adjunctive PMT among non-randomized, OCD-affected families receiving family-based group CBT. Linear mixed models estimated treatment effects across OCD-related and parenting outcomes at post-treatment and 1-month follow-up. Treatment response for 37 families receiving CBT + PMT (Mage = 13.90) was compared to 80 families receiving only CBT (Mage = 13.93). CBT + PMT was highly accepted by families. Families who received CBT + PMT had improved disruptive behaviors, parental distress tolerance, and other OCD-related outcomes. OCD-related outcomes did not significantly differ between groups. Results support CBT + PMT as effective treatment for pediatric OCD that may not provide incremental benefits beyond CBT alone. Future research should determine feasible and effective ways to incorporate key PMT components into CBT-based interventions.

Post-transcriptional Regulatory Functions of Mammalian Pumilio Proteins.

Mammalian Pumilio proteins, PUM1 and PUM2, are members of the PUF family of sequence-specific RNA-binding proteins. In this review, we explore their mechanisms, regulatory networks, biological functions, and relevance to diseases. Pumilio proteins bind an extensive network of mRNAs and repress protein expression by inhibiting translation and promoting mRNA decay. Opposingly, in certain contexts, they can activate protein expression. Pumilio proteins also regulate noncoding (nc)RNAs. The ncRNA, ncRNA activated by DNA damage (NORAD), can in turn modulate Pumilio activity. Genetic analysis provides new insights into Pumilio protein function. They are essential for growth and development. They control diverse processes, including stem cell fate, and neurological functions, such as behavior and memory formation. Novel findings show that their dysfunction contributes to neurodegeneration, epilepsy, movement disorders, intellectual disability, infertility, and cancer.

Binding synergy as an essential step for tRNA editing and modification enzyme codependence in Trypanosoma brucei.

Transfer RNAs acquire a variety of naturally occurring chemical modifications during their maturation; these fine-tune their structure and decoding properties in a manner critical for protein synthesis. We recently reported that in the eukaryotic parasite, Trypanosoma brucei, a methylation and deamination event are unexpectedly interconnected, whereby the tRNA adenosine deaminase (TbADAT2/3) and the 3-methylcytosine methyltransferase (TbTrm140) strictly rely on each other for activity, leading to formation of m3C and m3U at position 32 in several tRNAs. Still however, it is not clear why these two enzymes, which work independently in other systems, are strictly codependent in T. brucei Here, we show that these enzymes exhibit binding synergism, or a mutual increase in binding affinity, that is more than the sum of the parts, when added together in a reaction. Although these enzymes interact directly with each other, tRNA binding assays using enzyme variants mutated in critical binding and catalytic sites indicate that the observed binding synergy stems from contributions from tRNA-binding domains distal to their active sites. These results provide a rationale for the known interactions of these proteins, while also speaking to the modulation of substrate specificity between seemingly unrelated enzymes. This information should be of value in furthering our understanding of how tRNA modification enzymes act together to regulate gene expression at the post-transcriptional level and provide a basis for the interdependence of such activities.

ATP induces protein arginine deiminase 2-dependent citrullination in mast cells through the P2X7 purinergic receptor.

Posttranslational modifications regulate physiology either by directly modulating protein function or by impacting immune recognition of self-proteins. Citrullination is a posttranslational modification formed by the conversion of arginine residues into the citrulline amino acid by protein arginine deiminase (PAD) family members. We have identified mast cells as a major source of the PAD2 enzyme. Activation of the P2X7 purinergic receptor (P2X7) by the inflammatory "danger" signal ATP induces PAD2 activity and robust protein citrullination. P2X7-mediated activation of PAD2 is sensitive to p38 MAPK and protein kinase C inhibitors, and PAD2 regulates the expression of the TNFR2, Adamts-9, and Rab6b transcripts in mast cells. Further, the PAD2 enzyme and its citrullinated substrate proteins are released from mast cells on activation with ATP. PAD2 expression is closely linked with inflammation in rheumatoid arthritis (RA) synovial tissue, and PAD2 and citrullinated proteins are found in the synovial fluid of RA patients. In addition, RA is associated with the development of autoantibodies to citrullinated self-proteins. Our results suggest that P2X7 activation of mast cells may play a role in inflammation by providing PAD2 and PAD2 substrates access to the extracellular space.

Heterologous prime-boost vaccination drives early maturation of HIV broadly neutralizing antibody precursors in humanized mice.

A protective HIV vaccine will likely need to induce broadly neutralizing antibodies (bnAbs). Vaccination with the germline-targeting immunogen eOD-GT8 60mer adjuvanted with AS01B was found to induce VRC01-class bnAb precursors in 97% of vaccine recipients in the IAVI G001 phase 1 clinical trial; however, heterologous boost immunizations with antigens more similar to the native glycoprotein will be required to induce bnAbs. Therefore, we designed core-g28v2 60mer, a nanoparticle immunogen to be used as a first boost after eOD-GT8 60mer priming. We found, using a humanized mouse model approximating human conditions of VRC01-class precursor B cell diversity, affinity, and frequency, that both protein- and mRNA-based heterologous prime-boost regimens induced VRC01-class antibodies that gained key mutations and bound to near-native HIV envelope trimers lacking the N276 glycan. We further showed that VRC01-class antibodies induced by mRNA-based regimens could neutralize pseudoviruses lacking the N276 glycan. These results demonstrated that heterologous boosting can drive maturation toward VRC01-class bnAb development and supported the initiation of the IAVI G002 phase 1 trial testing mRNA-encoded nanoparticle prime-boost regimens.

Chemi-Northern: a versatile chemiluminescent northern blot method for analysis and quantitation of RNA molecules.

This report describes a chemiluminescence-based detection method for RNAs on northern blots, designated Chemi-Northern. This approach builds on the simplicity and versatility of northern blotting, while dispensing of the need for expensive and cumbersome radioactivity. RNAs are first separated on denaturing gel electrophoresis, transferred to a nylon membrane, and then hybridized to a biotinylated RNA or DNA antisense probe. Streptavidin conjugated with horseradish peroxidase and enhanced chemiluminescence substrate are then used to detect the probe bound to the target RNA. Our results demonstrate the versatility of this method in detecting natural and engineered RNAs expressed in cells, including messenger and noncoding RNAs. We show that Chemi-Northern detection is sensitive and fast, detecting attomole amounts of RNA in as little as 1 second, with high signal intensity and low background. The dynamic response displays excellent linearity. Using Chemi-Northern, we measure the significant, reproducible reduction of mRNA levels by human sequence-specific RNA-binding proteins, PUM1 and PUM2. Additionally, we measure the interaction of endogenous poly(A) binding protein, PABPC1, with poly-adenylated mRNA. Thus, the Chemi-Northern method provides a versatile, simple, cost-effective method to enable researchers to detect and measure changes in RNA expression, processing, binding, and decay of RNAs.

Effector function does not contribute to protection from virus challenge by a highly potent HIV broadly neutralizing antibody in nonhuman primates.

Protection from immunodeficiency virus challenge in nonhuman primates (NHPs) by a first-generation HIV broadly neutralizing antibody (bnAb) b12 has previously been shown to benefit from interaction between the bnAb and Fcγ receptors (FcγRs) on immune cells. To investigate the mechanism of protection for a more potent second-generation bnAb currently in clinical trials, PGT121, we carried out a series of NHP studies. These studies included treating with PGT121 at a concentration at which only half of the animals were protected to avoid potential masking of FcγR effector function benefits by dominant neutralization and using a new variant that more completely eliminated all rhesus FcγR binding than earlier variants. In contrast to b12, which required FcγR binding for optimal protection, we concluded that PGT121-mediated protection is not augmented by FcγR interaction. Thus, for HIV-passive antibody prophylaxis, these results, together with existing literature, emphasize the importance of neutralization potency for clinical antibodies, with effector function requiring evaluation for individual antibodies.

TbUTP10, a protein involved in early stages of pre-18S rRNA processing in Trypanosoma brucei.

Ribosome biosynthesis, best studied in opisthokonts, is a highly complex process involving numerous protein and RNA factors. Yet, very little is known about the early stages of pre-18S rRNA processing even in these model organisms, let alone the conservation of this mechanism in other eukaryotes. Here we extend our knowledge of this process by identifying and characterizing the essential protein TbUTP10, a homolog of yeast U3 small nucleolar RNA-associated protein 10 - UTP10 (HEATR1 in human), in the excavate parasitic protist Trypanosoma brucei. We show that TbUTP10 localizes to the nucleolus and that its ablation by RNAi knock-down in two different T. brucei life cycle stages results in similar phenotypes: a disruption of pre-18S rRNA processing, exemplified by the accumulation of rRNA precursors, a reduction of mature 18S rRNA, and also a decrease in the level of U3 snoRNA. Moreover, polysome profiles of the RNAi-induced knock-down cells show a complete disappearance of the 40S ribosomal subunit, and a prominent accumulation of the 60S large ribosomal subunit, reflecting impaired ribosome assembly. Thus, TbUTP10 is an important protein in the processing of 18S rRNA.

Group family-based cognitive behavioral therapy for pediatric obsessive compulsive disorder: Global outcomes and predictors of improvement.

This open, uncontrolled study examined the efficacy of a group family-based cognitive behavioral therapy (GF-CBT) protocol in treating pediatric obsessive-compulsive disorder (OCD) and explored predictors of symptom improvement. Eighty-five OCD-affected youth aged 8-18 years (M = 13.9 years, SD = 2.49; 46% male) and their parent(s) participated in a weekly, 12-session GF-CBT program. Data from multiple perspectives were gathered at the beginning and end of treatment, as well as at one-month follow-up. A broad range of assessment measures were utilized to capture clinically-relevant domains and a number of potential predictor variables were explored. Paired t-tests indicated that treatment was associated with significant reductions in clinician- and parent-rated OCD severity (d = 1.47, 1.32), youth and parent-rated functional impairment (d = 0.87, 0.67), coercive/disruptive behaviors (d = 0.75), and family accommodation (d = 1.02), as well as improvements in youth-, mother-, and father-rated family functioning (d = 1.05, 0.50, 0.88). Paired t-tests also indicated that youth remained improved at one-month follow-up. Step-wise regression identified greater homework success as a significant predictor of symptom improvement. This study provides evidence that GF-CBT significantly improves a wide range of domains for youth/families that extends beyond OCD symptom severity and supports homework as a core treatment component.

A case-control study of sleep disturbances in pediatric obsessive-compulsive disorder.

BACKGROUND: Sleep disturbances, including delayed sleep phase disorder (DSPD) and disorders of sleep initiation and maintenance (DIMS), have repeatedly been identified in adult obsessive-compulsive disorder (OCD). These disturbances have not been well-characterized objectively in pediatric OCD. METHODS: Thirty OCD-affected youth (8-18 yrs, 40% male) and 30 age and gender-matched healthy controls (HCs) completed the Sleep Disturbances Scale for Children (SDSC), and one week of continuous actigraphy with concurrent sleep diary documentation. A subsample completed the Children's Sleep Hygiene Scale (CSHS) and Sleep Attitudes and Beliefs Scale (SABS). RESULTS: Seventy-two percent of OCD participants reported sleep disturbances versus 15% of HC participants (p < 0.001). Convergent actigraphy results suggested DIMS but not DSPD were common. DISCUSSION: The parents of OCD-affected children seem to be successfully controlling bedtimes, preventing circadian rhythm system disruptions. OCD status does adversely impact, however, the perisleep arousal system. These results have important clinical implications, suggesting sleep problems may be best managed through direct treatment of OCD symptoms. It is recommended that all pediatric OCD patients be screened for sleep disturbances to inform treatment plan development.

The Evolution of Substrate Specificity by tRNA Modification Enzymes.

All types of nucleic acids in cells undergo naturally occurring chemical modifications, including DNA, rRNA, mRNA, snRNA, and most prominently tRNA. Over 100 different modifications have been described and every position in the purine and pyrimidine bases can be modified; often the sugar is also modified [1]. In tRNA, the function of modifications varies; some modulate global and/or local RNA structure, and others directly impact decoding and may be essential for viability. Whichever the case, the overall importance of modifications is highlighted by both their evolutionary conservation and the fact that organisms use a substantial portion of their genomes to encode modification enzymes, far exceeding what is needed for the de novo synthesis of the canonical nucleotides themselves [2]. Although some modifications occur at exactly the same nucleotide position in tRNAs from the three domains of life, many can be found at various positions in a particular tRNA and their location may vary between and within different tRNAs. With this wild array of chemical diversity and substrate specificities, one of the big challenges in the tRNA modification field has been to better understand at a molecular level the modes of substrate recognition by the different modification enzymes; in this realm RNA binding rests at the heart of the problem. This chapter will focus on several examples of modification enzymes where their mode of RNA binding is well understood; from these, we will try to draw general conclusions and highlight growing themes that may be applicable to the RNA modification field at large.

Prevalence of Acute-Onset Subtypes in Pediatric Obsessive-Compulsive Disorder.

BACKGROUND: Pediatric obsessive-compulsive disorder (OCD) is a common, debilitating illness. When childhood OCD symptom onset is described as acute and severe, diagnostic criteria for pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections (PANDAS) and pediatric acute-onset neuropsychiatric syndrome (PANS) should be considered. However, the frequency and differentiating features of these putative syndromes within pediatric OCD remain poorly understood. OBJECTIVES: To determine the prevalence and characteristics of those meeting PANDAS and/or PANS criteria within pediatric OCD, as determined by parent report and clinician interview. METHODS: Consecutive youth presenting to a subspecialty pediatric OCD clinic were rigorously assessed through the Anxiety Disorders Interview Schedule for DSM-IV, the Children's Yale-Brown Obsessive-Compulsive Scale, and through self- and parent-report measures, including a medical questionnaire. Strict diagnostic criteria for PANDAS and PANS were applied to determine prevalence rates, and comparative analyses were performed between subgroups. RESULTS: Among 136 youth with a lifetime OCD diagnosis, 5% (n = 7; 95% adjusted Wald interval: 1%-10%) met proposed criteria for PANDAS and/or PANS, of whom two met PANDAS criteria, four met PANS criteria, and one met criteria for both. Those in the PANDAS/PANS subgroup were more likely to have autoimmune illness, less likely to report symmetry factor symptoms, and had greater OCD-related family impairment during their worst OCD episode. CONCLUSION: A small yet significant percentage of pediatric OCD outpatients met criteria for PANDAS and/or PANS, justifying routine screening and attention to related characteristics during assessment and management. Longitudinal studies of these putative subtypes are warranted.

From Prebiotics to Probiotics: The Evolution and Functions of tRNA Modifications.

All nucleic acids in cells are subject to post-transcriptional chemical modifications. These are catalyzed by a myriad of enzymes with exquisite specificity and that utilize an often-exotic array of chemical substrates. In no molecule are modifications more prevalent than in transfer RNAs. In the present document, we will attempt to take a chemical rollercoaster ride from prebiotic times to the present, with nucleoside modifications as key players and tRNA as the centerpiece that drove the evolution of biological systems to where we are today. These ideas will be put forth while touching on several examples of tRNA modification enzymes and their modus operandi in cells. In passing, we submit that the choice of tRNA is not a whimsical one but rather highlights its critical function as an essential invention for the evolution of protein enzymes.

A case report of obsessive-compulsive disorder following acute disseminated encephalomyelitis.

We present a case of a boy who developed obsessive-compulsive disorder (OCD) shortly after an episode of acute disseminated encephalomyelitis (ADEM). To our knowledge, this is the first report of the development of OCD in a child who has had ADEM. This presentation is consistent with our understanding of OCD as a complex genetic disease involving the cerebral white matter tracts, and may indicate a potential pathway for the development of OCD in genetically vulnerable individuals or a shared trigger for the development of pediatric acute-onset neuropsychiatric syndrome and ADEM.

Conflict negotiation and autonomy processes in adolescent romantic relationships: an observational study of interdependency in boyfriend and girlfriend effects.

This study examined the association between conflict negotiation and the expression of autonomy in adolescent romantic partners. Thirty-seven couples participated in a globally coded conflict interaction task. Actor-partner interdependence models (APIM) were used to quantify the extent to which boys' and girls' autonomy was linked solely to their own negotiation of the conflict or whether it was linked conjointly to their own and their partners' negotiation style. Combining agentic autonomy theories and peer socialization models, it was expected that boys' and girls' autonomy would be associated only with their own conflict behaviors when they employed conflict styles reflective of their same gender repertoire, and associated conjointly with self and partner behaviors when they employed gender-atypical conflict styles. Instead of an equal, albeit distinct, positioning in the autonomy dynamic, the results suggested that girls' autonomy is associated solely with their own conflict behaviors, whereas boys' autonomy is jointly associated with their own and their partners' conflict behaviors. We discuss the relative power of boys and girls in emergent dyadic contexts, emphasizing how romantic dynamics shape salient abilities.