SARS-CoV-2 envelope protein triggers depression-like behaviors and dysosmia via TLR2-mediated neuroinflammation in mice.

BACKGROUND: Depression and dysosmia have been regarded as primary neurological symptoms in COVID-19 patients, the mechanism of which remains unclear. Current studies have demonstrated that the SARS-CoV-2 envelope (E) protein is a pro-inflammatory factor sensed by Toll-like receptor 2 (TLR2), suggesting the pathological feature of E protein is independent of viral infection. In this study, we aim to ascertain the role of E protein in depression, dysosmia and associated neuroinflammation in the central nervous system (CNS). METHODS: Depression-like behaviors and olfactory function were observed in both female and male mice receiving intracisternal injection of E protein. Immunohistochemistry was applied in conjunction with RT-PCR to evaluate glial activation, blood-brain barrier status and mediators synthesis in the cortex, hippocampus and olfactory bulb. TLR2 was pharmacologically blocked to determine its role in E protein-related depression-like behaviors and dysosmia in mice. RESULTS: Intracisternal injection of E protein evoked depression-like behaviors and dysosmia in both female and male mice. Immunohistochemistry suggested that the E protein upregulated IBA1 and GFAP in the cortex, hippocampus and olfactory bulb, while ZO-1 was downregulated. Moreover, IL-1ß, TNF-α, IL-6, CCL2, MMP2 and CSF1 were upregulated in both cortex and hippocampus, whereas IL-1ß, IL-6 and CCL2 were upregulated in the olfactory bulb. Furtherly, inhibiting microglia, rather than astrocytes, alleviated depression-like behaviors and dysosmia induced by E protein. Finally, RT-PCR and immunohistochemistry suggested that TLR2 was upregulated in the cortex, hippocampus and olfactory bulb, the blocking of which mitigated depression-like behaviors and dysosmia induced by E protein. CONCLUSIONS: Our study demonstrates that envelope protein could directly induce depression-like behaviors, dysosmia, and obvious neuroinflammation in CNS. TLR2 mediated depression-like behaviors and dysosmia induced by envelope protein, which could serve as a promising therapeutic target for neurological manifestation in COVID-19 patients.

C3aR in astrocytes mediates post-thoracotomy pain by inducing A1 astrocytes in male rats.

BACKGROUND: Astrocyte activation, which is polarized into classical neurotoxic A1, neuroprotective A2, A-pan, etc., is thought to be involved in the transition from acute to chronic post-thoracotomy pain. The C3aR receptor associated with astrocyte-neuron and -microglia interactions is necessary for A1 astrocytes polarization. This study aimed to determine whether C3aR in astrocytes mediates post-thoracotomy pain by inducing A1 expression in a rat thoracotomy pain model. METHODS: A rat thoracotomy pain model was employed. The mechanical withdraw threshold was measured to evaluate pain behavior. Lipopolysaccharide (LPS) was injected intraperitoneally to induce A1. Intrathecal injection of AAV2/9-rC3ar1 shRNA-GFAP was used to knock down in vivo C3aR expression in astrocytes. The expression of associated phenotypic markers before and after intervention was assessed by RT-PCR, western blot, co-immunofluorescence, and single-cell RNA sequencing. RESULTS: C3aR downregulation was found to inhibit LPS-induced A1 astrocytes activation, decrease the expression of C3aR, C3, and GFAP, which were activated from acute to chronic pain, and alleviate the mechanical withdrawal threshold and chronic pain incidence. In addition, more A2 astrocytes were activated in the model group that did not develop chronic pain. C3aR downregulation increased the number of A2 astrocytes upon LPS exposure. Knockdown of C3aR also decreased the activation of M1 microglia induced by LPS or thoracotomy. CONCLUSIONS: Our study confirmed that C3aR-induced A1 polarization contributes to chronic post-thoracotomy pain. Inhibition of A1 activation via C3aR downregulation increases anti-inflammatory A2 and decreases pro-inflammatory M1 activation, which may also be involved in the mechanism of chronic post-thoracotomy pain.

New ketamine analogue: 2-fluorodeschloro-N-ethyl-ketamine and its suggested metabolites.

Identifying new psychoactive substances (NPSs) and their metabolites is essential for regulating such substances for purposes of law enforcement and forensics. NPSs can be regulated on the basis of their chemical structures before they become a critical threat to society. Further, NPS metabolites can be targeted for analysis in urine, blood, and hair. This case study reports an incident in which 10 bags with approximately 15 g of crystalline material were seized from suspects by law enforcement officers and sent to the laboratory for confirmation. Gas chromatography-mass spectrometry, liquid chromatography-high-resolution mass spectrometry, and nuclear magnetic resonance (NMR) were employed to analyze these materials. The analyses revealed that the materials were a new ketamine analog, 2-fluorodeschloro-N-ethyl-ketamine (2-FDCNEK). Single-crystal X-ray diffraction (SXRD) analysis was also employed to confirm this result. In addition, metabolites of 2-FDCNEK were investigated using a fungal model and a urine sample from an abuser. The results suggest that 2-FDCNEK and 2-fluorodeschoro-norketamine are optimal metabolites for biological samples. This report presents the mass fragmentation, NMR analysis, and SXRD data of 2-FDCNEK. In addition, it provides suggestions regarding metabolites of 2-FDCNEK for law enforcement and forensic purposes, thereby facilitating the detection of this new ketamine analog.

Rich Get Richer: Extraversion Statistically Predicts Reduced Internet Addiction through Less Online Anonymity Preference and Extraversion Compensation.

Internet addiction may arise from multiple factors and personality tendencies have been previously implicated. Prior studies have found that extraversion may be a protective factor mitigating against internet addiction, yielding a "rich-get-richer" effect. However, few studies have explored how extraversion may influence internet addiction from the perspective of online-offline integration. Drawing on a sample of 428 college students, the current study examined a serial mediation model exploring the underlying mechanisms of how extraversion may statistically predict internet addiction through online-offline integration and antecedent factors. The serial mediation model analyses indicated that extraverted internet users exhibited a weaker preference for online anonymity and less online extraversion compensation, thus formulating a higher level of online-offline integration than introverted individuals, which, in turn, appeared to reduce the risk of internet addiction. In contrast, with regard to specific components of online-offline integration, introverted internet users preferred online anonymity, which reduced their relationship integration and increased their likelihood of internet addiction; similarly, the introverted individuals were also more likely to exhibit an extraversion compensation effect. That is, they were more extraverted on the internet than in general; hence, they had a lower level of self-identity integration, resulting in a greater likelihood of experiencing internet addiction. These results highlight the importance of online-offline integration that may account for personality variations in social and psychological outcomes related to internet use, and suggest a role for online anonymity preference and extraversion compensation in influencing specific components of integration.

Proteome Profile of Trigeminal Ganglion in Murine Model of Allergic Contact Dermatitis: Complement 3 Pathway Contributes to Itch and Pain Sensation.

Allergic contact dermatitis (ACD) is a common inflammatory dermatosis characterized by persistent itch and pain after topical contact with reactive chemicals. Although it has been long recognized as a type-IV hypersensitivity, its complexity of pathophysiology mechanism makes it still a clinical aporia in treatment. In this study, we aimed to identify crucial proteins involved in the nociceptive sensation of ACD. Based on a chemical-induced ACD murine model, we collected trigeminal ganglions of ACD and control mice for quantitative tandem mass tag (TMT)-labeling proteomic analysis. Immunohistochemistry was further practiced to validate the bioinformatic analysis. A total of 7685 proteins were identified and analyzed. Sixty-four proteins were significantly upregulated, and 75 proteins were downregulated in ACD mice. GO analysis demonstrated that the changed proteins were significantly enriched in terms of immune and peptidase activity in ACD mice. Proteins involved in the complement and coagulation cascades were notably changed in the KEGG enrichment analysis. The upregulation of complement component 3 (C3) in trigeminal satellite cells of ACD mice was further confirmed by immunohistochemistry. ACD upregulated C3 in trigeminal satellite cells. The complement system in sensory ganglion might play an essential role in forming pruritic and nociceptive sensations in ACD.

Lack of Spinal Neuropeptide Y Is Involved in Mechanical Itch in Aged Mice.

Neuropeptide Y (NPY) signaling plays an essential role in gating the pruritic afferent information in the spinal cord. Recent studies revealed that the aging process down-regulated the expression of NPY in the central nervous system. We propose that the lack of spinal NPY may be involved in certain types of pruritus in the elderly population. This study was designed to investigate the role of NPY in aging-induced itch using the senile mouse model. The expression of NPY in the spinal dorsal horn was compared between young (2 months old) and aged (24 months old) mice. Western blotting and immunohistochemistry showed that the expression of NPY was significantly reduced in the spinal dorsal horn in aged mice. In addition, a neuronal maker of apoptosis, TUNEL, was detected in the NPY positive neurons only in the aged spinal cord. Behavioral assay indicated that light mechanical stimulus evoked significantly more scratching in the aged than in the young mice, whereas chemical-evoked itch and pain-related behaviors were not altered. Intrathecal injection of either NPY or LP-NPY, a NPY receptor 1 (NPY1R) agonist, significantly alleviated the mechanically evoked itch in aged mice without altering the responses to chemical pruritogens. Our study suggested that downregulation of spinal NPY in the aged mice might play a role in the higher incidence of the mechanically evoked itch than that in the young mice. Therapies targeting the NPY system might serve as a potential strategy for alleviating the pruritic symptoms among the elderly population.

Suppression of TLR4-MyD88 signaling pathway attenuated chronic mechanical pain in a rat model of endometriosis.

BACKGROUND: As a classic innate immunity pathway, Toll-like receptor 4 (TLR4) signaling has been intensively investigated for its function of pathogen recognition. The receptor is located not only on immune cells but also on sensory neurons and spinal glia. Recent studies revealed the involvement of neuronal TLR4 in different types of pain. However, the specific role of TLR4 signaling in the pain symptom of endometriosis (EM) remains obscure. METHODS: The rat endometriosis model was established by transplanting uterine horn tissue into gastrocnemius. Western blotting and/or immunofluorescent staining were applied to detect high mobility group box 1 (HMGB1), TLR4, myeloid differentiation factor-88 adaptor protein (MyD88), and nuclear factor kappa-B-p65 (NF-κB-p65) expression, as well as the activation of astrocyte and microglia. The antagonist of TLR4 (LPS-RS-Ultra, LRU) and MyD88 homodimerization inhibitory peptide (MIP) were intrathecally administrated to assess the behavioral effects of blocking TLR4 signaling on endometriosis-related pain. RESULTS: Mechanical hyperalgesia was observed at the graft site, while HMGB1 was upregulated in the implanted uterine tissue, dorsal root ganglion (DRG), and spinal dorsal horn (SDH). Compared with sham group, upregulated TLR4, MyD88, and phosphorylated NF-κB-p65 were detected in the DRG and SDH in EM rats. The activation of astrocytes and microglia in the SDH was also confirmed in EM rats. Intrathecal application of LRU and MIP alleviated mechanical pain on the graft site of EM rats, with decreased phosphorylation of NF-κB-p65 in the DRG and reduced activation of glia in the SDH. CONCLUSIONS: HMGB1-TLR4-MyD88 signaling pathway in the DRG and SDH may involve in endometriosis-related hyperpathia. Blockade of TLR4 and MyD88 might serve as a potential treatment for pain in endometriosis.

CXCL12/CXCR4 signaling induced itch and pain sensation in a murine model of allergic contact dermatitis.

Allergic contact dermatitis is a skin inflammatory disease manifested with itch and pain symptom around the inflamed area. Chemokines such as CXCL12 are involved in the pathophysiology of allergic contact dermatitis, but little has been known about the effect of CXCL12/CXCR4 signaling for nociceptive sensation accompanying allergic contact dermatitis. Our study showed that CXCL12 and CXCR4 were upregulated in trigeminal ganglion with the progression of allergic contact dermatitis through western blotting and immunofluorescence. CXCL12 and CXCR4 were mainly upregulated in small-diameter neurons, which were co-localized with nociceptive markers in trigeminal ganglion. CXCR4 and CXCL12 were also expressed in trigeminal ganglion neurons retrograded from the skin lesion. Intradermal injection of CXCL12 enhanced the itch- and pain-like behavior which could be relieved by AMD3100, a CXCR4 antagonist, without changes of mast cells. Our findings suggested that blockade of CXCL12/CXCR4 signaling pathway might be beneficial to relieve itch and pain sensation accompanying allergic contact dermatitis.

Gender Moderates the Partial Mediation of Impulsivity in the Relationship Between Psychiatric Distress and Problematic Online Gaming: Online Survey.

BACKGROUND: Research has shown that some individuals can develop problematic patterns of online gaming, leading to significant psychological and interpersonal problems. Psychiatric distress and impulsivity have been suggested to contribute to problematic online gaming (POG). OBJECTIVE: This study aimed to investigate the potential mediating or moderating mechanisms of impulsivity and gender-related differences in possible associations between psychiatric distress and POG. METHODS: A total of 596 matched female and male participants, ranging in age from 14 to 38 years (mean 21.4, SD 4.5), were chosen from a large cross-sectional, nationwide Hungarian online gaming sample. Participants completed online questionnaires about self-reported impulsivity, psychiatric distress, and POG. RESULTS: Psychiatric distress directly predicted POG, and impulsivity partially mediated the relationship between psychiatric distress and POG. However, this mediation effect was found only for the impatience factor of impulsivity. Impulsivity did not moderate the relationship between psychiatric distress and POG. A moderating effect of gender was not found in the direct relationship between psychiatric distress and POG. However, a moderated mediation analysis revealed that impatience mediated the association between psychiatric distress and POG in males, whereas the indirect effect of impatience was not significant in females. CONCLUSIONS: The results of this work highlight gender-related difference among online gamers in the mediation effect of impulsivity between psychiatric distress and POG and provide novel insights regarding clinical implications for preventing or treating POG.

Corrigendum: Development of an Online and Offline Integration Hypothesis for Healthy Internet Use: Theory and Preliminary Evidence.

[This corrects the article DOI: 10.3389/fpsyg.2018.00492.].

The relationship between sexual sensation seeking and problematic Internet pornography use: A moderated mediation model examining roles of online sexual activities and the third-person effect.

BACKGROUND AND AIMS: Internet pornography consumption is prevalent among college students and problematic for some, yet little is known regarding the psychological constructs underlying problematic Internet pornography use (PIPU). Drawing on the Interaction of Person-Affect-Cognition-Execution model, this study tested a model that sexual sensation seeking (SSS) would impact PIPU through online sexual activities (OSAs) and that this relationship would be influenced by the third-person effect (TPE; a social cognitive bias relating to perceived impacts on others as compared to oneself) in a gender-sensitive manner. METHODS: A total of 808 Chinese college students (age range: 17-22 years, 57.7% male) were recruited and surveyed. RESULTS: Men scored higher than women on OSAs and PIPU and on each scale's component factors. The relationship between SSS and PIPU was mediated by OSAs, and the TPE moderated this relationship: the predictive path (SSS to PIPU) was significant only in participants with high TPE. The moderated mediation model was not invariant across gender groups, with data suggesting that it accounted for a greater proportion of the variance in men as compared with women. DISCUSSION AND CONCLUSIONS: The findings suggest that SSS may operate through participation in OSAs to lead to PIPU, and this relationship is particularly relevant for college-aged males scoring high on the TPE. These findings have implications for individuals who might be particularly vulnerable to developing PIPU and for guiding educational efforts and targeting interventions in college-aged students. The extent to which these findings extend to other age groups and cultures warrants further examination.

Development of an Online and Offline Integration Hypothesis for Healthy Internet Use: Theory and Preliminary Evidence.

The Internet has become an integral part of our daily life, and how to make the best use of the Internet is important to both individuals and the society. Based on previous studies, an Online and Offline Integration Hypothesis is proposed to suggest a framework for considering harmonious and balanced Internet use. The Integration Hypothesis proposes that healthier patterns of Internet usage may be achieved through harmonious integration of people's online and offline worlds. An online/offline integration is proposed to unite self-identity, interpersonal relationships, and social functioning with both cognitive and behavioral aspects by following the principles of communication, transfer, consistency, and "offline-first" priorities. To begin to test the hypothesis regarding the relationship between integration level and psychological outcomes, data for the present study were collected from 626 undergraduate students (41.5% males). Participants completed scales for online and offline integration, Internet addiction, pros and cons of Internet use, loneliness, extraversion, and life satisfaction. The findings revealed that subjects with higher level of online/offline integration have higher life satisfaction, greater extraversion, and more positive perceptions of the Internet and less loneliness, lower Internet addiction, and fewer negative perceptions of the Internet. Integration mediates the link between extraversion and psychological outcomes, and it may be the mechanism underlying the difference between the "rich get richer" and social compensation hypotheses. The implications of the online and offline integration hypothesis are discussed.

Efficacy assessment of an inactivated Tembusu virus vaccine candidate in ducks.

Duck Tembusu virus (TMUV) is a recently identified pathogen that causes severe egg drop and neurological disease in domestic duck and goose flocks. The infection has spread across the China mainland since its outbreak in 2010. Effective vaccines are needed to fight the disease. In this work, we describe the development and laboratory assessment of a cell culture-derived, inactivated duck TMUV vaccine. The TMUV-JXSP strain was successfully propagated on a baby hamster kidney cell line (BHK-21), inactivated with beta-propiolactone (BPL) and emulsified with mineral oil. The efficacy of different vaccination schedules was assessed in laying ducks and table ducks using virus challenge experiments. Two doses of vaccine provided efficient protection against the virus challenge to avoid the egg production drop in laying ducks. An ELISA demonstrated that 97% (39/40) of ducks seroconverted on day 21 after one dose of the inactivated vaccine and that significant increases in antibody titers against the virus were induced after the second immunization. For table ducks, a single dose of vaccine immunization resulted in a protection index of 87% and significant reduction of viral loads in tissues. Sterilizing immunity can be attained after second immunization. Our results demonstrate that BHK-21 cell culture is suitable for duck TMUV propagation and that BPL-inactivated TMUV vaccine can provide a high level of protection from virus challenge in laying ducks and table ducks. These data provide a scientific basis for the development of an inactivated vaccine for the prevention of duck TMUV infection.

Duck Tembusu virus exhibits neurovirulence in BALB/c mice.

BACKGROUND: Duck Tembusu virus is a member of the Ntaya group in the genus Flavivirus. The virus has been responsible for severe duck egg-drop syndrome in China since 2010. Its emergence and rapid spread have caused great economic loss for the poultry industry. The epidemiology of the virus infection and the potential threat to public health is of great concern because of the infective and zoonotic nature of flaviviruses. RESULTS: In this study, the pathogenicity of duck Tembusu virus in BALB/c mice was investigated. Infected mice developed clinical signs, including loss of appetite, ruffled hair, weight loss, disorientation, blindness and paralysis of hind limbs from six days post- infection following intracerebral inoculation. Morbidity was 100%, with mortality ranging from 20 to 80% in three- to eight-week-old mice. High virus titers were recovered from the brain, and the virus was distributed in several organs. Histologically, there was widespread non-suppurative encephalitis in the brain. Lymphocyte depletion in the spleen was observed, along with fatty degeneration in the liver and kidney. CONCLUSIONS: Our results demonstrate, for the first time, that duck Tembusu virus is highly neurovirulent in BALB/c mice. The mouse model used in this work was able to produce Tembusu virus infection and could be useful for elucidating some of the aspects of the pathophysiology of other flavivirus infections.

An infectious full-length cDNA clone of duck Tembusu virus, a newly emerging flavivirus causing duck egg drop syndrome in China.

Duck Tembusu virus (TMUV) is a recently identified pathogenic flavivirus that causes severe egg drop and encephalitis in Chinese ducks and geese. It has been found to be most closely related to the mosquito-origin Tembusu virus and chicken Sitiawan virus reported in Malaysia. However, the ecological characteristics and the pathogenesis of duck TMUV are largely unknown. We report the construction of full-length cDNA clone of duck TMUV strain JXSP. The virus genome was reverse transcribed, amplified as seven overlapping fragments and successively ligated into the low copy number vector pWSK29 under the control of a T7 promoter. Transfection of BHK-21 cells with the transcribed RNA from the full-length cDNA clone resulted in production of highly infectious progeny virus. In vitro growth characteristics in BHK-21 cells and virulence in ducklings and BALB/c mice were similar for the rescued and parental viruses. This stable infectious cDNA clone will be a valuable tool for studying the genetic determinants of duck TMUV.

Duck egg-drop syndrome caused by BYD virus, a new Tembusu-related flavivirus.

Since April 2010, a severe outbreak of duck viral infection, with egg drop, feed uptake decline and ovary-oviduct disease, has spread around the major duck-producing regions in China. A new virus, named BYD virus, was isolated in different areas, and a similar disease was reproduced in healthy egg-producing ducks, infecting with the isolated virus. The virus was re-isolated from the affected ducks and replicated well in primary duck embryo fibroblasts and Vero cells, causing the cytopathic effect. The virus was identified as an enveloped positive-stranded RNA virus with a size of approximately 55 nm in diameter. Genomic sequencing of the isolated virus revealed that it is closely related to Tembusu virus (a mosquito-borne Ntaya group flavivirus), with 87-91% nucleotide identity of the partial E (envelope) proteins to that of Tembusu virus and 72% of the entire genome coding sequence with Bagaza virus, the most closely related flavivirus with an entirely sequenced genome. Collectively our systematic studies fulfill Koch's postulates, and therefore, the causative agent of the duck egg drop syndrome occurring in China is a new flavivirus. Flavivirus is an emerging and re-emerging zoonotic pathogen and BYD virus that causes severe egg-drop, could be disastrous for the duck industry. More importantly its public health concerns should also be evaluated, and its epidemiology should be closely watched due to the zoonotic nature of flaviviruses.

Internet-based intervention for the treatment of online addiction for college students in China: a pilot study of the Healthy Online Self-helping Center.

Internet addiction among college students has become a serious problem in China. This pilot study involved the development of an online expert system named Healthy Online Self-helping Center (HOSC) as an intervention tool to help those who wish to reduce online usage. The study also explored the effectiveness of HOSC for college students' Internet addiction behavior. Participants (N = 65) were recruited from a university in Beijing, and were randomly assigned to one of four conditions: using HOSC within a laboratory environment, using HOSC within a natural environment, using a noninteractive program, and a control group. All the participants were asked to answer questionnaires at the baseline and at the 1-month follow-up. The questionnaires included the participants' online hours per week, the legitimate ratio of Internet usage, online satisfaction, and the Young's Diagnostic Questionnaire. The results revealed that HOSC under both natural and laboratory environments could effectively reduce the participants' online hours per week as well as their Young's Diagnostic Questionnaire score, and improve online satisfaction at a 1-month follow-up. Participants using a noninteractive program also had similar results. The article concludes with a discussion of the limitations of the study, as well as the implications of the findings and future research directions.

Isolation and characterization of a reovirus causing spleen necrosis in Pekin ducklings.

High rates of mortality for Pekin ducklings have been recorded in several duck farms in China since 2006. Dead ducklings were characterized by spleen necrosis, suggesting microbial infection as a cause of disease. Laboratory investigations led to the isolation of a virus strain from the spleen tissues of dead ducklings, designated DRV-HC. Subsequent experimental infections with DRV-HC resulted in marked spleen necrosis in the ducklings similar to those observed in the natural outbreaks. Electron microscopy of the cultured DRV-HC revealed viral particles that were non-enveloped and icosahedral with a mean diameter of approximately 72 nm. Agar gel precipitating tests showed that the isolate shared a common group-specific antigen with chicken reovirus S1133. DNA sequencing revealed that this isolate was closely related to Muscovy duck reoviruses. Experimental infection with DRV-HC resulted in death of young chicks with necrotic foci in the liver and spleen. To the best of our knowledge, this is the first report of the isolation of a duck reovirus with high virulence in Pekin ducklings and SPF chickens.