Altered medial prefrontal cortex and dorsal raphé activity predict genotype and correlate with abnormal learning behavior in a mouse model of autism-associated 2p16.3 deletion.

2p16.3 deletion, involving NEUREXIN1 (NRXN1) heterozygous deletion, substantially increases the risk of developing autism and other neurodevelopmental disorders. We have a poor understanding of how NRXN1 heterozygosity impacts on brain function and cognition to increase the risk of developing the disorder. Here we characterize the impact of Nrxn1α heterozygosity on cerebral metabolism, in mice, using 14 C-2-deoxyglucose imaging. We also assess performance in an olfactory-based discrimination and reversal learning (OB-DaRL) task and locomotor activity. We use decision tree classifiers to test the predictive relationship between cerebral metabolism and Nrxn1α genotype. Our data show that Nrxn1α heterozygosity induces prefrontal cortex (medial prelimbic cortex, mPrL) hypometabolism and a contrasting dorsal raphé nucleus (DRN) hypermetabolism. Metabolism in these regions allows for the predictive classification of Nrxn1α genotype. Consistent with reduced mPrL glucose utilization, prefrontal cortex insulin receptor signaling is decreased in Nrxn1α+/- mice. Behaviorally, Nrxn1α+/- mice show enhanced learning of a novel discrimination, impaired reversal learning and an increased latency to make correct choices. In addition, male Nrxn1α+/- mice show hyperlocomotor activity. Correlative analysis suggests that mPrL hypometabolism contributes to the enhanced novel odor discrimination seen in Nrxn1α+/- mice, while DRN hypermetabolism contributes to their increased latency in making correct choices. The data show that Nrxn1α heterozygosity impacts on prefrontal cortex and serotonin system function, which contribute to the cognitive alterations seen in these animals. The data suggest that Nrxn1α+/- mice provide a translational model for the cognitive and behavioral alterations seen in autism and other neurodevelopmental disorders associated with 2p16.3 deletion. LAY SUMMARY: Deletion of the chromosomal region 2p16.3, involving reduced NEUREXIN1 gene expression, dramatically increases the risk of developing autism. Here, we show that reduced Neurexin1α expression, in mice, impacts on the prefrontal cortex and impairs cognitive flexibility. The data suggest that 2p16.3 deletion increases the risk of developing autism by impacting on the prefrontal cortex. Mice with the deletion are a useful model for testing new drugs to treat the cognitive flexibility problems experienced by people with autism.

Immunogenicity and protective efficacy of an intranasal live-attenuated vaccine against SARS-CoV-2.

Global deployment of an effective and safe vaccine is necessary to curtail the coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we evaluated a Newcastle disease virus (NDV)-based vectored-vaccine in mice and hamsters for its immunogenicity, safety, and protective efficacy against SARS-CoV-2. Intranasal administration of recombinant (r)NDV-S vaccine expressing spike (S) protein of SARS-CoV-2 to mice induced high levels of SARS-CoV-2-specific neutralizing immunoglobulin A (IgA) and IgG2a antibodies and T-cell-mediated immunity. Hamsters immunized with two doses of vaccine showed complete protection from lung infection, inflammation, and pathological lesions following SARS-CoV-2 challenge. Importantly, administration of two doses of intranasal rNDV-S vaccine significantly reduced the SARS-CoV-2 shedding in nasal turbinate and lungs in hamsters. Collectively, intranasal vaccination has the potential to control infection at the site of inoculation, which should prevent both clinical disease and virus transmission to halt the spread of the COVID-19 pandemic.

Artificial Intelligence-Assisted Loop Mediated Isothermal Amplification (AI-LAMP) for Rapid Detection of SARS-CoV-2.

Until vaccines and effective therapeutics become available, the practical solution to transit safely out of the current coronavirus disease 19 (CoVID-19) lockdown may include the implementation of an effective testing, tracing and tracking system. However, this requires a reliable and clinically validated diagnostic platform for the sensitive and specific identification of SARS-CoV-2. Here, we report on the development of a de novo, high-resolution and comparative genomics guided reverse-transcribed loop-mediated isothermal amplification (LAMP) assay. To further enhance the assay performance and to remove any subjectivity associated with operator interpretation of results, we engineered a novel hand-held smart diagnostic device. The robust diagnostic device was further furnished with automated image acquisition and processing algorithms and the collated data was processed through artificial intelligence (AI) pipelines to further reduce the assay run time and the subjectivity of the colorimetric LAMP detection. This advanced AI algorithm-implemented LAMP (ai-LAMP) assay, targeting the RNA-dependent RNA polymerase gene, showed high analytical sensitivity and specificity for SARS-CoV-2. A total of ~200 coronavirus disease (CoVID-19)-suspected NHS patient samples were tested using the platform and it was shown to be reliable, highly specific and significantly more sensitive than the current gold standard qRT-PCR. Therefore, this system could provide an efficient and cost-effective platform to detect SARS-CoV-2 in resource-limited laboratories.

Ketamine Restores Thalamic-Prefrontal Cortex Functional Connectivity in a Mouse Model of Neurodevelopmental Disorder-Associated 2p16.3 Deletion.

2p16.3 deletions, involving heterozygous NEUREXIN1 (NRXN1) deletion, dramatically increase the risk of developing neurodevelopmental disorders, including autism and schizophrenia. We have little understanding of how NRXN1 heterozygosity increases the risk of developing these disorders, particularly in terms of the impact on brain and neurotransmitter system function and brain network connectivity. Thus, here we characterize cerebral metabolism and functional brain network connectivity in Nrxn1α heterozygous mice (Nrxn1α+/- mice), and assess the impact of ketamine and dextro-amphetamine on cerebral metabolism in these animals. We show that heterozygous Nrxn1α deletion alters cerebral metabolism in neural systems implicated in autism and schizophrenia including the thalamus, mesolimbic system, and select cortical regions. Nrxn1α heterozygosity also reduces the efficiency of functional brain networks, through lost thalamic "rich club" and prefrontal cortex (PFC) hub connectivity and through reduced thalamic-PFC and thalamic "rich club" regional interconnectivity. Subanesthetic ketamine administration normalizes the thalamic hypermetabolism and partially normalizes thalamic disconnectivity present in Nrxn1α+/- mice, while cerebral metabolic responses to dextro-amphetamine are unaltered. The data provide new insight into the systems-level impact of heterozygous Nrxn1α deletion and how this increases the risk of developing neurodevelopmental disorders. The data also suggest that the thalamic dysfunction induced by heterozygous Nrxn1α deletion may be NMDA receptor-dependent.

ILC2s mediate systemic innate protection by priming mucus production at distal mucosal sites.

Host immunity to parasitic nematodes requires the generation of a robust type 2 cytokine response, characterized by the production of interleukin 13 (IL-13), which drives expulsion. Here, we show that infection with helminths in the intestine also induces an ILC2-driven, IL-13-dependent goblet cell hyperplasia and increased production of mucins (Muc5b and Muc5ac) at distal sites, including the lungs and other mucosal barrier sites. Critically, we show that type 2 priming of lung tissue through increased mucin production inhibits the progression of a subsequent lung migratory helminth infection and limits its transit through the airways. These data show that infection by gastrointestinal-dwelling helminths induces a systemic innate mucin response that primes peripheral barrier sites for protection against subsequent secondary helminth infections. These data suggest that innate-driven priming of mucus barriers may have evolved to protect from subsequent infections with multiple helminth species, which occur naturally in endemic areas.

Intestinal epithelial suppressor of cytokine signaling 3 (SOCS3) impacts on mucosal homeostasis in a model of chronic inflammation.

INTRODUCTION: Suppressor of cytokine signaling 3 (SOCS3) is a tumour suppressor, limiting intestinal epithelial cell (IEC) proliferation in acute inflammation, and tumour growth, but little is known regarding its role in mucosal homeostasis. Resistance to the intestinal helminth Trichuris muris relies on an "epithelial escalator" to expel the parasite. IEC turnover is restricted by parasite-induced indoleamine 2,3-dioxygenase (IDO). METHODS: Mice with or without conditional knockout of SOCS3 were infected with T. muris. Crypt depth, worm burden, and proliferating cells and IDO were quantified. SOCS3 knockdown was also performed in human IEC cell lines. RESULTS: Chronic T. muris infection increased expression of SOCS3 in wild-type mice. Lack of IEC SOCS3 led to a modest increase in epithelial turnover. This translated to a lower worm burden, but not complete elimination of the parasite suggesting a compensatory mechanism, possibly IDO, as seen in SOCS3 knockdown. CONCLUSIONS: We report that SOCS3 impacts on IEC turnover following T. muris infection, potentially through enhancement of IDO. IDO may dampen the immune response which can drive IEC hyperproliferation in the absence of SOCS3, demonstrating the intricate interplay of immune signals regulating mucosal homeostasis, and suggesting a novel tumour suppressor role of SOCS3.

The Differential Hormonal Milieu of Morning versus Evening May Have an Impact on Muscle Hypertrophic Potential.

Substantial gains in muscle strength and hypertrophy are clearly associated with the routine performance of resistance training. What is less evident is the optimal timing of the resistance training stimulus to elicit these significant functional and structural skeletal muscle changes. Therefore, this investigation determined the impact of a single bout of resistance training performed either in the morning or evening upon acute anabolic signalling (insulin-like growth factor-binding protein-3 (IGFBP-3), myogenic index and differentiation) and catabolic processes (cortisol). Twenty-four male participants (age 21.4±1.9yrs, mass 83.7±13.7kg) with no sustained resistance training experience were allocated to a resistance exercise group (REP). Sixteen of the 24 participants were randomly selected to perform an additional non-exercising control group (CP) protocol. REP performed two bouts of resistance exercise (80% 1RM) in the morning (AM: 0800 hrs) and evening (PM: 1800 hrs), with the sessions separated by a minimum of 72 hours. Venous blood was collected immediately prior to, and 5 min after, each resistance exercise and control sessions. Serum cortisol and IGFBP-3 levels, myogenic index, myotube width, were determined at each sampling period. All data are reported as mean ± SEM, statistical significance was set at P≤0.05. As expected a significant reduction in evening cortisol concentration was observed at pre (AM: 98.4±10.5, PM: 49.8±4.4 ng/ml, P<0.001) and post (AM: 98.0±9.0, PM: 52.7±6.0 ng/ml, P<0.001) exercise. Interestingly, individual cortisol differences pre vs post exercise indicate a time-of-day effect (AM difference: -2±2.6%, PM difference: 14.0±6.7%, P = 0.03). A time-of-day related elevation in serum IGFBP-3 (AM: 3274.9 ± 345.2, PM: 3605.1 ± 367.5, p = 0.032) was also evident. Pre exercise myogenic index (AM: 8.0±0.6%, PM: 16.8±1.1%) and myotube width (AM: 48.0±3.0, PM: 71.6±1.9 µm) were significantly elevated (P<0.001) in the evening. Post exercise myogenic index was greater AM (11.5±1.6%) compared with PM (4.6±0.9%). No difference was observed in myotube width (AM: 48.5±1.5, PM: 47.8±1.8 µm) (P>0.05). Timing of resistance training regimen in the evening appears to augment some markers of hypertrophic potential, with elevated IGFBP-3, suppressed cortisol and a superior cellular environment. Further investigation, to further elucidate the time course of peak anabolic signalling in morning vs evening training conditions, are timely.

Listeria monocytogenes alters mast cell phenotype, mediator and osteopontin secretion in a listeriolysin-dependent manner.

Whilst mast cells participate in the immune defence against the intracellular bacterium Listeria monocytogenes, there is conflicting evidence regarding the ability of L. monocytogenes to infect mast cells. It is known that the pore-forming toxin listeriolysin (LLO) is important for mast cell activation, degranulation and the release of pro-inflammatory cytokines. Mast cells, however, are a potential source of a wide range of cytokines, chemokines and other mediators including osteopontin, which contributes to the clearing of L. monocytogenes infections in vivo, although its source is unknown. We therefore aimed to resolve the controversy of mast cell infection by L. monocytogenes and investigated the extent of mediator release in response to the bacterium. In this paper we show that the infection of bone marrow-derived mast cells by L. monocytogenes is inefficient and LLO-independent. LLO, however, is required for calcium-independent mast cell degranulation as well as for the transient and selective downregulation of cell surface CD117 (c-kit) on mast cells. We demonstrate that in addition to the key pro-inflammatory cytokines TNF-α and IL-6, mast cells release a wide range of other mediators in response to L. monocytogenes. Osteopontin, IL-2, IL-4, IL-13 and granulocyte macrophage colony-stimulating factor (GM-CSF), and chemokines including CCL2, CCL3, CCL4 and CCL5 are released in a MyD88-dependent manner. The wide range of mediators released by mast cells in response to L. monocytogenes may play an important role in the recruitment and activation of a variety of immune cells in vivo. The cocktail of mediators, however, is unlikely to skew the immune response to a particular effector response. We propose that mast cells provide a hitherto unreported source of osteopontin, and may provide an important role in co-ordinating the immune response during Listeria infection.

IL-33 causes selective mast cell tolerance to bacterial cell wall products by inducing IRAK1 degradation.

Mast cells are important cellular constituents of epithelial-mesenchymal interactions, densely located at sites of microbial entry into the host where they are continuously exposed to products from commensals. In order to avoid excessive activation and the associated pathology, mast cell responses to TLR agonists must be tightly regulated. Here, we show that exposure in vitro to subactivating levels of the epithelial cell product, IL-33, renders mast cells insensitive to bacterial cell wall products. Mast cell responsiveness to Ag, cytoplasmic dsDNA, and TLR7/8 agonists is unaffected or enhanced by IL-33. The IL-33-induced mast cell selective tolerance requires the IL-33 receptor ST2 and peritoneal mast cells from St2(-/-) mice display a constitutively activated phenotype, demonstrated by increased expression of activation markers including CD11b and CD28. IL-33 exposure neither affects the levels of TLR4, MyD88, TIRAP, IL-1R associated kinase 2 (IRAK2), or IRAK4, nor induces persistent A20 or Tollip expression, but potently causes ST2-dependent IRAK1 degradation. We show that while IRAK2 is redundant for TLR4 signaling, IRAK1 is essential for TLR4 signaling in mast cells. We suggest that IL-33 produced during homeostasis retains mast cells in an unresponsive state to bacterial cell wall products via IRAK1 degradation, thus preventing chronic inflammation and tissue destruction.

PPARγ as a molecular target of EPA anti-inflammatory activity during TNF-α-impaired skeletal muscle cell differentiation.

Activated skeletal muscle satellite cells facilitate muscle repair or growth through proliferation, differentiation and fusion into new or existing myotubes. Elevated levels of the proinflammatory cytokine tumor necrosis factor-α (TNF-α) impair this process and are documented to have significant roles in muscle pathology. Recent evidence shows that the ω-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA) can block TNF-mediated suppression of progenitor cell differentiation, but the nature of this activity and its significance for local regulation of inflammation are not known. In the current study, we examined differentiation of the C2C12 myoblast line during treatment with TNF-α and EPA and measured the expression, activation and inhibition of peroxisome proliferator-activated receptor-γ (PPARγ), as several studies have shown its involvement in mediating EPA activity and the inhibition of nuclear factor (NF)-κB inflammatory gene activation. We found that TNF-α treatment increased NF-κB activity and reduced expression and activation of PPARγ, resulting in impaired myotube formation. EPA treatment attenuated these effects of TNF-α and was associated with up-regulation of PPARγ. Furthermore, EPA inhibited TNF-α-mediated transcription and secretion of interleukin (IL)-6, a key target gene of TNF-mediated NF-κB transcriptional activity. Pretreatment with a PPARγ selective antagonist inhibited some of the actions of EPA but was only partially effective in reversing inhibition of IL-6 production. These results show that EPA activity was associated with altered expression and activation of PPARγ, but exerted through both PPARγ-dependent and PPARγ-independent pathways leading to suppression of the proinflammatory cellular microenvironment.