MLN4924 Inhibits Defective Ribosomal Product Antigen Presentation Independently of Direct NEDDylation of Protein Antigens.

Successful direct MHC class I Ag presentation is dependent on the protein degradation machinery of the cell to generate antigenic peptides that can be loaded onto MHC class I molecules for surveillance by CD8+ T cells of the immune system. Most often this process involves the ubiquitin (Ub)-proteasome system; however, other Ub-like proteins have also been implicated in protein degradation and direct Ag presentation. In this article, we examine the role of neuronal precursor cell-expressed developmentally downregulated protein 8 (NEDD8) in direct Ag presentation in mouse cells. NEDD8 is the Ub-like protein with highest similarity to Ub, and fusion of NEDD8 to the N terminus of a target protein can lead to the degradation of target proteins. We find that appending NEDD8 to the N terminus of the model Ag OVA resulted in degradation by both the proteasome and the autophagy protein degradation pathways, but only proteasomal degradation, involving the proteasomal subunit NEDD8 ultimate buster 1, resulted in peptide presentation. When directly compared with Ub, NEDD8 fusion was less efficient at generating peptides. However, inactivation of the NEDD8-conugation machinery by treating cells with MLN4924 inhibited the presentation of peptides from the defective ribosomal product-derived form of a model Ag. These results demonstrate that NEDD8 activity in the cell is important for direct Ag presentation, but not by directly targeting proteins for degradation.

Inhibition of the Deubiquitinase Usp14 Diminishes Direct MHC Class I Antigen Presentation.

Infected or transformed cells must present peptides derived from endogenous proteins on MHC class I molecules to be recognized and targeted for elimination by Ag-specific cytotoxic T cells. In the first step of peptide generation, proteins are degraded by the proteasome. In this study, we investigated the role of the ubiquitin-specific protease 14 (Usp14), a proteasome-associated deubiquitinase, in direct Ag presentation using a ligand-stabilized model protein expressed as a self-antigen. Chemical inhibition of Usp14 diminished direct presentation of the model antigenic peptide, and the effect was especially pronounced when presentation was restricted to the defective ribosomal product (DRiP) form of the protein. Additionally, presentation specifically from DRiP Ags was diminished by expression of a catalytically inactive form of Usp14. Usp14 inhibition did not appreciably alter protein synthesis and only partially delayed protein degradation as measured by a slight increase in the half-life of the model protein when its degradation was induced. Taken together, these data indicate that functional Usp14 enhances direct Ag presentation, preferentially of DRiP-derived peptides, suggesting that the processing of DRiPs is in some ways different from other forms of Ag.

A neurocomputational investigation of reinforcement-based decision making as a candidate latent vulnerability mechanism in maltreated children.

Alterations in reinforcement-based decision making may be associated with increased psychiatric vulnerability in children who have experienced maltreatment. A probabilistic passive avoidance task and a model-based functional magnetic resonance imaging analytic approach were implemented to assess the neurocomputational components underlying decision making: (a) reinforcement expectancies (the representation of the outcomes associated with a stimulus) and (b) prediction error signaling (the ability to detect the differences between expected and actual outcomes). There were three main findings. First, the maltreated group (n = 18; mean age = 13), relative to nonmaltreated peers (n = 19; mean age = 13), showed decreased activity during expected value processing in a widespread network commonly associated with reinforcement expectancies representation, including the striatum (especially the caudate), the orbitofrontal cortex, and medial temporal structures including the hippocampus and insula. Second, consistent with previously reported hyperresponsiveness to negative cues in the context of childhood abuse, the maltreated group showed increased prediction error signaling in the middle cingulate gyrus, somatosensory cortex, superior temporal gyrus, and thalamus. Third, the maltreated group showed increased activity in frontodorsal regions and in the putamen during expected value representation. These findings suggest that early adverse environments disrupt the development of decision-making processes, which in turn may compromise psychosocial functioning in ways that increase latent vulnerability to psychiatric disorder.

Enhanced Direct Major Histocompatibility Complex Class I Self-Antigen Presentation Induced by Chlamydia Infection.

The direct major histocompatibility complex (MHC) class I antigen presentation pathway ensures intracellular peptides are displayed at the cellular surface for recognition of infected or transformed cells by CD8(+) cytotoxic T lymphocytes. Chlamydia spp. are obligate intracellular bacteria and, as such, should be targeted by CD8(+) T cells. It is likely that Chlamydia spp. have evolved mechanisms to avoid the CD8(+) killer T cell responses by interfering with MHC class I antigen presentation. Using a model system of self-peptide presentation which allows for posttranslational control of the model protein's stability, we tested the ability of various Chlamydia species to alter direct MHC class I antigen presentation. Infection of the JY lymphoblastoid cell line limited the accumulation of a model host protein and increased presentation of the model-protein-derived peptides. Enhanced self-peptide presentation was detected only when presentation was restricted to defective ribosomal products, or DRiPs, and total MHC class I levels remained unaltered. Skewed antigen presentation was dependent on a bacterial synthesized component, as evidenced by reversal of the observed phenotype upon preventing bacterial transcription, translation, and the inhibition of bacterial lipooligosaccharide synthesis. These data suggest that Chlamydia spp. have evolved to alter the host antigen presentation machinery to favor presentation of defective and rapidly degraded forms of self-antigen, possibly as a mechanism to diminish the presentation of peptides derived from bacterial proteins.

The sexually dimorphic impact of maltreatment on cortical thickness, surface area and gyrification.

An extensive literature has detailed how maltreatment experience impacts brain structure in children and adolescents. However, there is a dearth of studies on the influence of maltreatment on surface based indices, and to date no study has investigated how sex influences the impact of maltreatment on cortical thickness, surface area and local gyrification. We investigated sex differences in these measures of cortical structure in a large community sample of children aged 10-14 years (n = 122) comprising 62 children with verified maltreatment experience and 60 matched non-maltreated controls. The maltreated group relative to the controls presented with a pattern of decreased cortical thickness within a region of right anterior cingulate, orbitofrontal cortex and superior frontal gyrus; decreased surface area within the right inferior parietal cortex; and increased local gyrification within left superior parietal cortex. This atypical pattern of cortical structure was similar across males and females. An interaction between maltreatment exposure and sex was found only in local gyrification, within two clusters: the right tempo-parietal junction and the left precentral gyrus. These findings suggest that maltreatment impacts cortical structure in brain areas associated with emotional regulation and theory of mind, with few differences between the sexes.

Sex differences in socioemotional functioning, attentional bias, and gray matter volume in maltreated children: A multilevel investigation.

While maltreatment is known to impact social and emotional functioning, threat processing, and neural structure, the potentially dimorphic influence of sex on these outcomes remains relatively understudied. We investigated sex differences across these domains in a large community sample of children aged 10 to 14 years (n = 122) comprising 62 children with verified maltreatment experience and 60 well-matched nonmaltreated peers. The maltreated group relative to the nonmaltreated comparison group exhibited poorer social and emotional functioning (more peer problems and heightened emotional reactivity). Cognitively, they displayed a pattern of attentional avoidance of threat in a visual dot-probe task. Similar patterns were observed in males and females in these domains. Reduced gray matter volume was found to characterize the maltreated group in the medial orbitofrontal cortex, bilateral middle temporal lobes, and bilateral supramarginal gyrus; sex differences were observed only in the supramarginal gyrus. In addition, a disordinal interaction between maltreatment exposure and sex was found in the postcentral gyrus. Finally, attentional avoidance to threat mediated the relationship between maltreatment and emotional reactivity, and medial orbitofrontal cortex gray matter volume mediated the relationship between maltreatment and peer functioning. Similar mediation patterns were observed across sexes. This study highlights the utility of combining multiple levels of analysis when studying the "latent vulnerability" engendered by childhood maltreatment and yields tentative findings regarding a neural basis of sex differences in long-term outcomes for maltreated children.

ROS-induced nanotherapeutic approach for ovarian cancer treatment based on the combinatorial effect of photodynamic therapy and DJ-1 gene suppression.

This study represents a novel approach for intraoperative ovarian cancer treatment based on the combinatorial effect of a targeted photodynamic therapy (PDT) associated with suppression of the DJ-1 protein, one of the key players in the ROS defense of cancer cells. To assess the potential of the developed therapy, dendrimer-based nanoplatforms for cancer-targeted delivery of near-infrared photosensitizer, phthalocyanine, and DJ-1 siRNA have been constructed. In vitro studies revealed that therapeutic efficacy of the combinatorial approach was enhanced when compared to PDT alone and this enhancement was more pronounced in ovarian carcinoma cells, which are characterized by higher basal levels of DJ-1 protein. Moreover, the ovarian cancer tumors exposed to a single dose of combinatorial therapy were completely eradicated from the mice and the treated animals showed no evidence of cancer recurrence. Thus, the developed therapeutic approach can be potentially employed intraoperatively to eradicate unresactable cancer cells. FROM THE CLINICAL EDITOR: The complete clearance of microscopic residual tumor cells during excision surgery is important to improve survival of the patient. In this interesting paper, the authors developed a novel approach using targeted photodynamic therapy (PDT), combining a photosensitizer, phthalocyanine, and DJ-1 siRNA for the treatment of ovarian cancer. The data showed that this approach increased cancer cell killing and may pave way for future clinical studies.

Mycobacterium abscessus infection results in decrease of oxidative metabolism of lung airways cells and relaxation of the epithelial mucosal tight junctions.

Mycobacterium abscessus complex is a group of environmental pathogens that recently have been isolated more from patients with underlying lung diseases, such and COPD, bronchiectasis, and cystic fibrosis. The mechanisms involved in the pathogenesis of these diseases have only recently been investigated. Infection is associated with biofilm formation on the airway mucosa, invasion of the mucosal epithelial cells and a time-dependent impairment of the integrity of the monolayer. Using electron microscopy, it was shown that Mycobacterium abscessus induced lesions of the cell surface structures. Tight junction proteins claudin-1 and occludin-1 have increased transcription in cells exposed to Mycobacterium abscessus, in contrast to cells exposed to Mycobacterium avium. Infection of A549 alveolar epithelial cells by Mycobacterium abscessus reduced the oxidative metabolism of the cell, without inducing necrosis. A transposon library screen identified mutants that do not alter the metabolism of the A549 cells.Once the bacterium crosses the epithelial barrier, it may encounter sub-epithelial macrophages. Select mutants were used for infection assays to determine their effects on membrane integrity. Translocated select mutants were attenuated in macrophages compared to wild type Mycobacterium abscessus. In summary, the dynamics of Mycobacterium abscessus infection appears to be different from other non-tuberculous mycobacteria (NTMs). Future studies will attempt to address the mechanism involved in airway membrane lesions.

Direct Conjugation of NEDD8 to the N-Terminus of a Model Protein Can Induce Degradation.

While the role of ubiquitin in protein degradation is well established, the role of other ubiquitin-like proteins (UBLs) in protein degradation is less clear. Neural precursor cell expressed developmentally down-regulated protein 8 (NEDD8) is the UBL with the highest level of amino acids identified when compared to ubiquitin. Here we tested if the N-terminal addition of NEDD8 to a protein of interest could lead to degradation. Mutation of critical glycine residues required for normal NEDD8 processing resulted in a non-cleavable fusion protein that was rapidly degraded within the cells by both the proteasome and autophagy. Both degradation pathways were dependent on a functional ubiquitin-conjugation system as treatment with MLN7243 increased levels of non-cleavable NEDD8-GFP. The degradation of non-cleavable, N-terminal NEDD8-GFP was not due to a failure of GFP folding as different NEDD8-GFP constructs with differing abilities to fold and fluoresce were similarly degraded. Though the fusion of NEDD8 to a protein resulted in degradation, treatment of cells with MLN4924, an inhibitor of the E1 activating enzyme for NEDD8, failed to prevent degradation of other destabilized substrates. Taken together these data suggest that under certain conditions, such as the model system described here, the covalent linkage of NEDD8 to a protein substrate may result in the target proteins degradation.

Mapping neural activity patterns to contextualized fearful facial expressions onto callous-unemotional (CU) traits: intersubject representational similarity analysis reveals less variation among high-CU adolescents.

Callous-unemotional (CU) traits are early-emerging personality features characterized by deficits in empathy, concern for others, and remorse following social transgressions. One of the interpersonal deficits most consistently associated with CU traits is impaired behavioral and neurophysiological responsiveness to fearful facial expressions. However, the facial expression paradigms traditionally employed in neuroimaging are often ambiguous with respect to the nature of threat (i.e., is the perceiver the threat, or is something else in the environment?). In the present study, 30 adolescents with varying CU traits viewed fearful facial expressions cued to three different contexts ("afraid for you," "afraid of you," "afraid for self") while undergoing functional magnetic resonance imaging (fMRI). Univariate analyses found that mean right amygdala activity during the "afraid for self" context was negatively associated with CU traits. With the goal of disentangling idiosyncratic stimulus-driven neural responses, we employed intersubject representational similarity analysis to link intersubject similarities in multivoxel neural response patterns to contextualized fearful expressions with differential intersubject models of CU traits. Among low-CU adolescents, neural response patterns while viewing fearful faces were most consistently similar early in the visual processing stream and among regions implicated in affective responding, but were more idiosyncratic as emotional face information moved up the cortical processing hierarchy. By contrast, high-CU adolescents' neural response patterns consistently aligned along the entire cortical hierarchy (but diverged among low-CU youths). Observed patterns varied across contexts, suggesting that interpretations of fearful expressions depend to an extent on neural response patterns and are further shaped by levels of CU traits.

Mycobacterium avium subsp. hominissuis effector MAVA5_06970 promotes rapid apoptosis in secondary-infected macrophages during cell-to-cell spread.

Mycobacterium avium subsp. hominissuis is an opportunistic intracellular pathogen associated with disease in patients either immunosuppression or chronic lung pathology. Once in the host, M. avium preferentially infects and replicates within the phagocytic cells. The host driven macrophage apoptosis appears to be an essential aspect of innate immunity during bacterial infection; however, the existing evidence suggests that M. avium has evolved adaptive approaches to trigger the phagocyte apoptosis, exit apoptotic cells or via ingestion of infected apoptotic bodies subsequently infect neighboring macrophages. By evaluating 4,000 transposon mutants of M. avium in THP-1 cells, we identified clones that can trigger a new form of early host cell apoptosis, which is only observed upon entry into the "secondary-infected" macrophages. Inactivation of MAVA5_06970 gene lead to significant attenuation in intracellular growth within macrophages and mice, and impaired M. avium to induce rapid apoptosis in the "secondary-infected" cells as measured by Annexin V-FITC detection assay. Complementation of MAVA5_06970 gene corrected the attenuation as well as apoptotic phenotypes. The MAVA5_06970 gene encodes for a secreted protein. Using the pull-down assay and then confirmed with the yeast two-hybrid screen, we found that MAVA5_06970 effector interacts with the Secreted Phosphoprotein 1, the cytokine also known as Osteopontin. This interaction enhances the THP-1 cell apoptosis and, consequently, restricts the production of interleukin-12 that likely may limit the activation of the type I immunity pathway in vivo. This work identified a key virulence effector of M. avium that contributes to the cell-to-cell spread of the pathogen.

Detection of bacterial-reactive natural IgM antibodies in desert bighorn sheep populations.

Ecoimmunology is a burgeoning field of ecology which studies immune responses in wildlife by utilizing general immune assays such as the detection of natural antibody. Unlike adaptive antibodies, natural antibodies are important in innate immune responses and often recognized conserved epitopes present in pathogens. Here, we describe a procedure for measuring natural antibodies reactive to bacterial antigens that may be applicable to a variety of organisms. IgM from desert bighorn sheep plasma samples was tested for reactivity to outer membrane proteins from Vibrio coralliilyticus, a marine bacterium to which sheep would have not been exposed. Immunoblotting demonstrated bighorn sheep IgM could bind to a variety of bacterial cell envelope proteins while ELISA analysis allowed for rapid determination of natural antibody levels in hundreds of individual animals. Natural antibody levels were correlated with the ability of plasma to kill laboratory strains of E. coli bacteria. Finally, we demonstrate that natural antibody levels varied in two distinct populations of desert bighorn sheep. These data demonstrate a novel and specific measure of natural antibody function and show that this varies in ecologically relevant ways.

The Inverse Relationship between the Microstructural Variability of Amygdala-Prefrontal Pathways and Trait Anxiety Is Moderated by Sex.

Anxiety impacts the quality of everyday life and may facilitate the development of affective disorders, possibly through concurrent alterations in neural circuitry. Findings from multimodal neuroimaging studies suggest that trait-anxious individuals may have a reduced capacity for efficient communication between the amygdala and the ventral prefrontal cortex (vPFC). A diffusion-weighted imaging protocol with 61 directions was used to identify lateral and medial amygdala-vPFC white matter pathways. The structural integrity of both pathways was inversely correlated with self-reported levels of trait anxiety. When this mask from our first dataset was then applied to an independent validation dataset, both pathways again showed a consistent inverse relationship with trait anxiety. Importantly, a moderating effect of sex was found, demonstrating that the observed brain-anxiety relationship was stronger in females. These data reveal a potential neuroanatomical mediator of previously documented functional alterations in amygdala-prefrontal connectivity that is associated with trait anxiety, which might prove informative for future studies of psychopathology.

MHC class I antigen presentation of DRiP-derived peptides from a model antigen is not dependent on the AAA ATPase p97.

CD8(+) T cells are responsible for killing cells of the body that have become infected or oncogenically transformed. In order to do so, effector CD8(+) T cells must recognize their cognate antigenic peptide bound to a MHC class I molecule that has been directly presented by the target cell. Due to the rapid nature of antigen presentation, it is believed that antigenic peptides are derived from a subset of newly synthesized proteins which are degraded almost immediately following synthesis and termed Defective Ribosomal Products or DRiPs. We have recently reported on a bioassay which can distinguish antigen presentation of DRiP substrates from other forms of rapidly degraded proteins and found that poly-ubiquitin chain disassembly may be necessary for efficient DRiP presentation. The AAA ATPase p97 protein is necessary for efficient cross-presentation of antigens on MHC class I molecules and plays an important role in extracting mis-folded proteins from the endoplasmic reticulum. Here, we find that genetic ablation or chemical inhibition of p97 does not diminish DRiP antigen presentation to any great extent nor does it alter the levels of MHC class I molecules on the cell surface, despite our observations that p97 inhibition increased the levels of poly-ubiquitinated proteins in the cell. These data demonstrate that inhibiting poly-ubiquitin chain disassembly alone is insufficient to abolish DRiP presentation.

Metabolism and pharmacokinetics of the anti-tuberculosis drug ethionamide in a flavin-containing monooxygenase null mouse.

Multiple drug resistance (MDR) in Mycobacterium tuberculosis (mTB), the causative agent for tuberculosis (TB), has led to increased use of second-line drugs, including ethionamide (ETA). ETA is a prodrug bioactivated by mycobacterial and mammalian flavin-containing monooxygenases (FMOs). FMO2 is the major isoform in the lungs of most mammals, including primates. In humans a polymorphism exists in the expression of FMO2. FMO2.2 (truncated, inactive) protein is produced by the common allele, while the ancestral allele, encoding active FMO2.1, has been documented only in individuals of African and Hispanic origin, at an incidence of up to 50% and 7%, respectively. We hypothesized that FMO2 variability in TB-infected individuals would yield differences in concentrations and ratios of ETA prodrug and metabolites. In this study we assessed the impact of the FMO2 genetic polymorphism on the pharmacokinetics of ETA after administration of a single oral dose of ETA (125 mg/kg) to wild type and triple Fmo1/2/4-null mice, measuring levels of prodrug vs. metabolites in plasma collected from 0 to 3.5 h post-gavage. All mice metabolized ETA to ETA S-oxide (ETASO) and 2-ethyl-4-amidopyridine (ETAA). Wild type mice had higher plasma concentrations of metabolites than of parent compound (p = 0.001). In contrast, Fmo1/2/4-null mice had higher plasma concentrations of parent compound than of metabolites (p = 0.0001). Thus, the human FMO2 genotype could impact the therapeutic efficacy and/or toxicity of ETA.

The structural and functional connectivity of the amygdala: from normal emotion to pathological anxiety.

The dynamic interactions between the amygdala and the medial prefrontal cortex (mPFC) are usefully conceptualized as a circuit that both allows us to react automatically to biologically relevant predictive stimuli as well as regulate these reactions when the situation calls for it. In this review, we will begin by discussing the role of this amygdala-mPFC circuitry in the conditioning and extinction of aversive learning in animals. We will then relate these data to emotional regulation paradigms in humans. Finally, we will consider how these processes are compromised in normal and pathological anxiety. We conclude that the capacity for efficient crosstalk between the amygdala and the mPFC, which is represented as the strength of the amygdala-mPFC circuitry, is crucial to beneficial outcomes in terms of reported anxiety.