Nonenveloped Avian Reoviruses Released with Small Extracellular Vesicles Are Highly Infectious.

Vesicle-encapsulated nonenveloped viruses are a recently recognized alternate form of nonenveloped viruses that can avoid immune detection and potentially increase systemic transmission. Avian orthoreoviruses (ARVs) are the leading cause of various disease conditions among birds and poultry. However, whether ARVs use cellular vesicle trafficking routes for egress and cell-to-cell transmission is still poorly understood. We demonstrated that fusogenic ARV-infected quail cells generated small (~100 nm diameter) extracellular vesicles (EVs) that contained electron-dense material when observed by transmission electron microscope. Cryo-EM tomography indicated that these vesicles did not contain ARV virions or core particles, but the EV fractions of OptiPrep gradients did contain a small percent of the ARV virions released from cells. Western blotting of detergent-treated EVs revealed that soluble virus proteins and the fusogenic p10 FAST protein were contained within the EVs. Notably, virus particles mixed with the EVs were up to 50 times more infectious than virions alone. These results suggest that EVs and perhaps fusogenic FAST-EVs could contribute to ARV virulence.

Kaposi's sarcoma-associated herpesvirus (KSHV) utilizes the NDP52/CALCOCO2 selective autophagy receptor to disassemble processing bodies.

Kaposi's sarcoma-associated herpesvirus (KSHV) causes the inflammatory and angiogenic endothelial cell neoplasm, Kaposi's sarcoma (KS). We previously demonstrated that the KSHV Kaposin B (KapB) protein promotes inflammation via the disassembly of cytoplasmic ribonucleoprotein granules called processing bodies (PBs). PBs modify gene expression by silencing or degrading labile messenger RNAs (mRNAs), including many transcripts that encode inflammatory or angiogenic proteins associated with KS disease. Although our work implicated PB disassembly as one of the causes of inflammation during KSHV infection, the precise mechanism used by KapB to elicit PB disassembly was unclear. Here we reveal a new connection between the degradative process of autophagy and PB disassembly. We show that both latent KSHV infection and KapB expression enhanced autophagic flux via phosphorylation of the autophagy regulatory protein, Beclin. KapB was necessary for this effect, as infection with a recombinant virus that does not express the KapB protein did not induce Beclin phosphorylation or autophagic flux. Moreover, we showed that PB disassembly mediated by KSHV or KapB, depended on autophagy genes and the selective autophagy receptor NDP52/CALCOCO2 and that the PB scaffolding protein, Pat1b, co-immunoprecipitated with NDP52. These studies reveal a new role for autophagy and the selective autophagy receptor NDP52 in promoting PB turnover and the concomitant synthesis of inflammatory molecules during KSHV infection.

DLL4 and VCAM1 enhance the emergence of T cell-competent hematopoietic progenitors from human pluripotent stem cells.

T cells show tremendous efficacy as cellular therapeutics. However, obtaining primary T cells from human donors is expensive and variable. Pluripotent stem cells (PSCs) have the potential to provide a renewable source of T cells, but differentiating PSCs into hematopoietic progenitors with T cell potential remains an important challenge. Here, we report an efficient serum- and feeder-free system for differentiating human PSCs into hematopoietic progenitors and T cells. This fully defined approach allowed us to study the impact of individual proteins on blood emergence and differentiation. Providing DLL4 and VCAM1 during the endothelial-to-hematopoietic transition enhanced downstream progenitor T cell output by ~80-fold. These two proteins synergized to activate notch signaling in nascent hematopoietic stem and progenitor cells, and VCAM1 additionally promoted an inflammatory transcriptional program. We also established optimized medium formulations that enabled efficient and chemically defined maturation of functional CD8αß+, CD4-, CD3+, TCRαß+ T cells with a diverse TCR repertoire.

Human coronaviruses disassemble processing bodies.

A dysregulated proinflammatory cytokine response is characteristic of severe coronavirus infections caused by SARS-CoV-2, yet our understanding of the underlying mechanism responsible for this imbalanced immune response remains incomplete. Processing bodies (PBs) are cytoplasmic membraneless ribonucleoprotein granules that control innate immune responses by mediating the constitutive decay or suppression of mRNA transcripts, including many that encode proinflammatory cytokines. PB formation promotes turnover or suppression of cytokine RNAs, whereas PB disassembly corresponds with the increased stability and/or translation of these cytokine RNAs. Many viruses cause PB disassembly, an event that can be viewed as a switch that rapidly relieves cytokine RNA repression and permits the infected cell to respond to viral infection. Prior to this submission, no information was known about how human coronaviruses (CoVs) impacted PBs. Here, we show SARS-CoV-2 and the common cold CoVs, OC43 and 229E, induced PB loss. We screened a SARS-CoV-2 gene library and identified that expression of the viral nucleocapsid (N) protein from SARS-CoV-2 was sufficient to mediate PB disassembly. RNA fluorescent in situ hybridization revealed that transcripts encoding TNF and IL-6 localized to PBs in control cells. PB loss correlated with the increased cytoplasmic localization of these transcripts in SARS-CoV-2 N protein-expressing cells. Ectopic expression of the N proteins from five other human coronaviruses (OC43, MERS, 229E, NL63 and SARS-CoV) did not cause significant PB disassembly, suggesting that this feature is unique to SARS-CoV-2 N protein. These data suggest that SARS-CoV-2-mediated PB disassembly contributes to the dysregulation of proinflammatory cytokine production observed during severe SARS-CoV-2 infection.

Viral Manipulation of a Mechanoresponsive Signaling Axis Disassembles Processing Bodies.

Processing bodies (PBs) are ribonucleoprotein granules important for cytokine mRNA decay that are targeted for disassembly by many viruses. Kaposi's sarcoma-associated herpesvirus is the etiological agent of the inflammatory endothelial cancer, Kaposi's sarcoma, and a PB-regulating virus. The virus encodes kaposin B (KapB), which induces actin stress fibers (SFs) and cell spindling as well as PB disassembly. We now show that KapB-mediated PB disassembly requires actin rearrangements, RhoA effectors, and the mechanoresponsive transcription activator, YAP. Moreover, ectopic expression of active YAP or exposure of ECs to mechanical forces caused PB disassembly in the absence of KapB. We propose that the viral protein KapB activates a mechanoresponsive signaling axis and links changes in cell shape and cytoskeletal structures to enhanced inflammatory molecule expression using PB disassembly. Our work implies that cytoskeletal changes in other pathologies may similarly impact the inflammatory environment.

Raloxifene prevents stress granule dissolution, impairs translational control and promotes cell death during hypoxia in glioblastoma cells.

Glioblastoma (GBM) is the most common primary malignant brain tumor, and it has a uniformly poor prognosis. Hypoxia is a feature of the GBM microenvironment, and previous work has shown that cancer cells residing in hypoxic regions resist treatment. Hypoxia can trigger the formation of stress granules (SGs), sites of mRNA triage that promote cell survival. A screen of 1120 FDA-approved drugs identified 129 candidates that delayed the dissolution of hypoxia-induced SGs following a return to normoxia. Amongst these candidates, the selective estrogen receptor modulator (SERM) raloxifene delayed SG dissolution in a dose-dependent manner. SG dissolution typically occurs by 15 min post-hypoxia, however pre-treatment of immortalized U251 and U3024 primary GBM cells with raloxifene prevented SG dissolution for up to 2 h. During this raloxifene-induced delay in SG dissolution, translational silencing was sustained, eIF2α remained phosphorylated and mTOR remained inactive. Despite its well-described role as a SERM, raloxifene-mediated delay in SG dissolution was unaffected by co-administration of ß-estradiol, nor did ß-estradiol alone have any effect on SGs. Importantly, the combination of raloxifene and hypoxia resulted in increased numbers of late apoptotic/necrotic cells. Raloxifene and hypoxia also demonstrated a block in late autophagy similar to the known autophagy inhibitor chloroquine (CQ). Genetic disruption of the SG-nucleating proteins G3BP1 and G3BP2 revealed that G3BP1 is required to sustain the raloxifene-mediated delay in SG dissolution. Together, these findings indicate that modulating the stress response can be used to exploit the hypoxic niche of GBM tumors, causing cell death by disrupting pro-survival stress responses and control of protein synthesis.

Null results of oxytocin and vasopressin administration across a range of social cognitive and behavioral paradigms: Evidence from a randomized controlled trial.

Research examining oxytocin and vasopressin in humans has the potential to elucidate neurobiological mechanisms underlying human sociality that have been previously unknown or not well characterized. A primary goal of this work is to increase our knowledge about neurodevelopmental and psychiatric disorders characterized by impairments in social cognition. However, years of research highlighting wide-ranging effects of, in particular, intranasal oxytocin administration have been tempered as the fields of psychology, neuroscience, and other disciplines have been addressing concerns over the reproducibility and validity of research findings. We present a series of behavioral tasks that were conducted using a randomized, double-blind, placebo controlled, between-subjects design, in which our research group found no main effects of oxytocin and vasopressin on a host of social outcomes. In addition to null hypothesis significance testing, we implemented equivalence testing and Bayesian hypothesis testing to examine the sensitivity of our findings. These analyses indicated that 47-83% of our results (depending on the method of post-hoc analysis) had enough sensitivity to detect the absence of a main effect. Our results add to evidence that intranasal oxytocin may have a more limited direct effect on human social processes than initially assumed and suggest that the direct effects of intranasal vasopressin may be similarly limited. Randomized controlled trial registration: NCT01680718.

Oxytocin, but not vasopressin, impairs social cognitive ability among individuals with higher levels of social anxiety: a randomized controlled trial.

Individuals with social anxiety are characterized by a high degree of social sensitivity, which can coincide with impairments in social cognitive functioning (e.g. theory of mind). Oxytocin (OT) and vasopressin (AVP) have been shown to improve social cognition, and OT has been theorized as a potential therapeutic agent for individuals with social anxiety disorder. However, no study has investigated whether these neuropeptides improve social cognitive ability among socially anxious individuals. In a randomized, double-blind, placebo controlled, between-subjects design we investigated whether social anxiety moderated the effects of OT or AVP (vs placebo) on social working memory (i.e. working memory that involves manipulating social information) and non-social working memory. OT vs placebo impaired social working memory accuracy in participants with higher levels of social anxiety. No differences were found for non-social working memory or for AVP vs placebo. Results suggest that OT administration in individuals with higher levels of social anxiety may impair social cognitive functioning. Randomized-controlled trial registration: NCT01680718.

Vasopressin, but not oxytocin, increases empathic concern among individuals who received higher levels of paternal warmth: A randomized controlled trial.

BACKGROUND: Empathy improves our ability to communicate in social interactions and motivates prosocial behavior. The neuropeptides arginine vasopressin and oxytocin play key roles in socioemotional processes such as pair bonding and parental care, which suggests that they may be involved in empathic processing. METHODS: We investigated how vasopressin and oxytocin affect empathic responding in a randomized, double-blind, placebo controlled, between-subjects study design. We also examined the moderating role of parental warmth, as reported in the early family environment, on empathic responding following vasopressin, oxytocin, or placebo administration. RESULTS: Among participants who reported higher levels of paternal warmth (but not maternal warmth), vasopressin (vs. placebo and oxytocin) increased ratings of empathic concern after viewing distressing and uplifting videos. No main or interaction effects were found for individuals who received oxytocin. CONCLUSIONS: Vasopressin has a role in enhancing empathy among individuals who received higher levels of paternal warmth. TRIAL REGISTRATION: NCT01680718.

Unraveling the anxious mind: anxiety, worry, and frontal engagement in sustained attention versus off-task processing.

Much remains unknown regarding the relationship between anxiety, worry, sustained attention, and frontal function. Here, we addressed this using a sustained attention task adapted for functional magnetic resonance imaging. Participants responded to presentation of simple stimuli, withholding responses to an infrequent "No Go" stimulus. Dorsolateral prefrontal cortex (DLPFC) activity to "Go" trials, and dorsal anterior cingulate (dACC) activity to "No Go" trials were associated with faster error-free performance; consistent with DLPFC and dACC facilitating proactive and reactive control, respectively. Trait anxiety was linked to reduced recruitment of these regions, slower error-free performance, and decreased frontal-thalamo-striatal connectivity. This indicates an association between trait anxiety and impoverished frontal control of attention, even when external distractors are absent. In task blocks where commission errors were made, greater DLPFC-precuneus and DLPFC-posterior cingulate connectivity were associated with both trait anxiety and worry, indicative of increased off-task thought. Notably, unlike trait anxiety, worry was not linked to reduced frontal-striatal-thalamo connectivity, impoverished frontal recruitment, or slowed responding during blocks without commission errors, contrary to accounts proposing a direct causal link between worry and impoverished attentional control. This leads us to propose a new model of the relationship between anxiety, worry and frontal engagement in attentional control versus off-task thought.

Moderate threat causes longer lasting disruption to processing in anxious individuals.

Anxiety is associated with increased attentional capture by threat. Previous studies have used simultaneous or briefly separated (<1 s) presentation of threat distractors and target stimuli. Here, we tested the hypothesis that high trait anxious participants would show a longer time window within which distractors cause disruption to subsequent task processing, and that this would particularly be observed for stimuli of moderate or ambiguous threat value. A novel temporally separated emotional distractor task was used. Face or house distractors were presented for 250 ms at short (∼1.6 s) or long (∼3 s) intervals prior to a letter string comprising Xs or Ns. Trait anxiety was associated with slowed identification of letter strings presented at long intervals after face distractors with part surprise/part fear expressions. In other words, these distractors had an impact on high anxious individuals' speed of target identification seconds after their offset. This was associated with increased activity in the fusiform gyrus and amygdala and reduced dorsal anterior cingulate recruitment. This pattern of activity may reflect impoverished recruitment of reactive control mechanisms to damp down stimulus-specific processing in subcortical and higher visual regions. These findings have implications for understanding how threat-related attentional biases in anxiety may lead to dysfunction in everyday settings where stimuli of moderate, potentially ambiguous, threat value such as those used here are fairly common, and where attentional disruption lasting several seconds may have a profound impact.

Neural and behavioral bases of age differences in perceptions of trust.

Older adults are disproportionately vulnerable to fraud, and federal agencies have speculated that excessive trust explains their greater vulnerability. Two studies, one behavioral and one using neuroimaging methodology, identified age differences in trust and their neural underpinnings. Older and younger adults rated faces high in trust cues similarly, but older adults perceived faces with cues to untrustworthiness to be significantly more trustworthy and approachable than younger adults. This age-related pattern was mirrored in neural activation to cues of trustworthiness. Whereas younger adults showed greater anterior insula activation to untrustworthy versus trustworthy faces, older adults showed muted activation of the anterior insula to untrustworthy faces. The insula has been shown to support interoceptive awareness that forms the basis of "gut feelings," which represent expected risk and predict risk-avoidant behavior. Thus, a diminished "gut" response to cues of untrustworthiness may partially underlie older adults' vulnerability to fraud.

An fMRI study of caring vs self-focus during induced compassion and pride.

This study examined neural activation during the experience of compassion, an emotion that orients people toward vulnerable others and prompts caregiving, and pride, a self-focused emotion that signals individual strength and heightened status. Functional magnetic resonance images (fMRI) were acquired as participants viewed 55 s continuous sequences of slides to induce either compassion or pride, presented in alternation with sequences of neutral slides. Emotion self-report data were collected after each slide condition within the fMRI scanner. Compassion induction was associated with activation in the midbrain periaqueductal gray (PAG), a region that is activated during pain and the perception of others' pain, and that has been implicated in parental nurturance behaviors. Pride induction engaged the posterior medial cortex, a region that has been associated with self-referent processing. Self-reports of compassion experience were correlated with increased activation in a region near the PAG, and in the right inferior frontal gyrus (IFG). Self-reports of pride experience, in contrast, were correlated with reduced activation in the IFG and the anterior insula. These results provide preliminary evidence towards understanding the neural correlates of important interpersonal dimensions of compassion and pride. Caring (compassion) and self-focus (pride) may represent core appraisals that differentiate the response profiles of many emotions.