The double-stranded RNA-dependent protein kinase PKR negatively regulates the protein expression of IFN-ß induced by RIG-I signaling.

Retinoic acid-inducible gene-I (RIG-I) is a cytoplasmic RNA sensor that plays an important role in innate immune responses to viral RNAs. Double-stranded RNA (dsRNA)-dependent protein kinase (PKR) is a eukaryotic initiation factor 2α (eIF2α) kinase that is initially involved in the responses of the translational machinery to dsRNA. PKR is also thought to play an essential role in antiviral innate immunity. However, the coordinated mechanisms of RIG-I and PKR that induce the expression of type I interferons (IFNs), essential cytokines involved in antiviral defense, are not completely understood. In this study, we show that PKR negatively participates in the RIG-I-mediated induction of IFN-ß expression. Stress granule (SG) formation is crucial to sequester mRNA to prevent aberrant protein synthesis by various stresses. SG formation in response to dsRNA was triggered by a PKR-mediated antiviral stress response. However, IFN-ß mRNA was not sequestered in the SGs of dsRNA-treated cells. dsRNA-induced translational silencing was thought to be PKR dependent. However, our results indicated that some proteins, including IFN-ß, were clearly translated despite PKR-mediated translational silencing. This study suggests that RIG-I responds mainly to IFN-ß expression in cells to which non-self dsRNA is introduced. In addition, PKR negatively regulates IFN-ß protein expression induced by RIG-I signaling. This may explain the essential role of PKR in fine-tuning the expression of IFN-ß in RIG-I-mediated antiviral immune responses.

Daily Brazilian green propolis intake elevates blood artepillin C levels in humans.

BACKGROUND: Propolis is a natural product collected by worker bees from a variety of plant species. As a type of propolis, Brazilian green propolis contains a large amount of artepillin C. Artepillin C is a cinnamic acid derivative and has been shown to have a wide variety of biological functions, including anti-inflammatory, antiviral and antitumor activities, in both cell culture and animal models. However, how propolis is digested and absorbed remains to be elucidated. Moreover, blood artepillin C levels after propolis intake have not been shown in human studies. RESULTS: A randomized, single-blind placebo-controlled study on the effect of Brazilian green propolis on serum artepillin C levels was conducted with healthy volunteers. The participants (n = 133) were randomly allocated in an approximately 2:1 ratio to two groups: propolis (n = 91) and placebo (n = 42). The participants took daily propolis or placebo, and blood tests were performed on day 0 (before propolis intake) and days 1, 3 and 7. Artepillin C was detected in serum in almost all individuals in the propolis groups. No serum artepillin C was detected in the placebo group. Serum artepillin C levels in the female group tended to be higher than those in the male group. In the female group, menstrual status was unrelated to serum artepillin C levels. CONCLUSION: These results suggested that propolis intake might be more effective for females than for males. © 2021 Society of Chemical Industry.

The Essential Role of Double-Stranded RNA-Dependent Antiviral Signaling in the Degradation of Nonself Single-Stranded RNA in Nonimmune Cells.

The recognition of nonself dsRNA by retinoic acid-inducible gene-I (RIG-I) leads to the engagement of RIG-I-like receptor signaling. In addition, nonself dsRNA triggers a robust latent RNase (RNase L) activation and leads to the degradation of ribosomal structures and cell death. In contrast, nonself ssRNA is known to be recognized by TLR 7/8 in immune cells such as plasmacytoid dendritic cells and B cells, but little is known regarding the involvement of nonself ssRNA in antiviral signaling in nonimmune cells, including epithelial cells. Moreover, the fate of intracellular nonself ssRNA remains unknown. To address this issue, we developed a quantitative RT-PCR-based approach that monitors the kinetics of nonself ssRNA cleavage following the transfection of HeLa human cervical carcinoma cells, using model nonself ssRNA. We discovered that the degradation of ssRNA is independent of RIG-I and type I IFN signaling because ssRNA did not trigger RIG-I-mediated antiviral signaling. We also found that the kinetics of self (5'-capped) and nonself ssRNA decay were unaltered, suggesting that nonself ssRNA is not recognized by nonimmune cells. We further demonstrated that the cleavage of nonself ssRNA is accelerated when nonself dsRNA is also introduced into cells. In addition, the cleavage of nonself ssRNA is completely abolished by knockdown of RNase L. Overall, our data demonstrate the important role of dsRNA-RNase L in nonself ssRNA degradation and may partly explain the positive regulation of the antiviral responses in nonimmune cells.

HSP90 Regulation of P2X7 Receptor Function Requires an Intact Cytoplasmic C-Terminus.

P2X7 receptors (P2X7Rs) are ATP-gated ion channels that display the unusual property of current facilitation during long applications of agonists. Here we show that facilitation disappears in chimeric P2X7Rs containing the C-terminus of the P2X2 receptor (P2X2R), and in a truncated P2X7R missing the cysteine-rich domain of the C-terminus. The chimeric and truncated receptors also show an apparent decreased permeability to N-methyl-d-glucamine(+) (NMDG(+)). The effects of genetic modification of the C-terminus on NMDG(+) permeability were mimicked by preapplication of the HSP90 antagonist geldanamycin to the wild-type receptor. Further, the geldanamycin decreased the shift in the reversal potential of the ATP-gated current measured under bi-ionic NMDG(+)/Na(+) condition without affecting the ability of the long application of agonist to facilitate current amplitude. Taken together, the results suggest that HSP90 may be essential for stabilization and function of P2X7Rs through an action on the cysteine-rich domain of the cytoplasmic the C-terminus.

ER export signals mediate plasma membrane localization of transmembrane protein TMEM72.

Transmembrane protein 72 (TMEM72) is involved in normal kidney development and tumorigenesis in renal cell carcinoma. However, the function of TMEM72 has not been experimentally examined; therefore, the role of TMEM72 is incompletely understood. In this study, we initially demonstrated that TMEM72 has four transmembrane domains (TMDs) and a long C-terminal tail. Immunofluorescence analysis showed that TMEM72 is localized on the plasma membrane but not on the outer mitochondrial membrane. Experiments performed with a series of TMEM72 deletion mutants and an evaluation of the unfolded protein response indicated that these TMDs are needed for proper protein folding or assembly. In contrast, domain-specific replacement analysis indicated the essential role of the C-terminal region of TMEM72 in protein transport. Spatial colocalization and immunoprecipitation assays showed that the proximal C-terminal region is responsible for anterograde protein transport. An amino acid sequence analysis and an immunocytochemical evaluation revealed that KRKKRKAAPEVLA, which corresponds to amino acid positions 132-144 in TMEM72, participates in efficient cellular transport. The motifs 132KRKKRK137 and 139APEVLA144 are associated with COPII and are considered to cooperate with membrane trafficking. Because efficient membrane trafficking is crucial for cells to maintain normal function, our data may contribute to elucidating the pathogenesis of membrane trafficking-associated diseases, particularly renal carcinoma and chronic kidney disease.

Appetite loss and associated factors at 1 year after intensive care unit elder survivors in a secondary analysis of the SMAP-HoPe study.

Appetite loss, a common but serious issue in older patients, is an independent risk factor for sarcopenia, which is associated with high mortality. However, few studies have explored the phenomenon of appetite loss after discharge from the intensive care unit (ICU). Therefore, we aimed to describe the prevalence of appetite loss and relationship between appetite loss and depression in patients living at home 12 months after intensive care. This study involved secondary analysis of data obtained from a published ambidirectional study examining post-intensive care syndrome 12 months after discharge (SMAP-HoPe study) conducted in 12 ICUs in Japan. We included patients aged > 65 years. The Short Nutritional Assessment Questionnaire and Hospital Anxiety Depression Scale were used for the analysis. Descriptive statistics and a multilevel generalized linear model were used to clarify the relationship between appetite loss and depression. Data from 468 patients were analyzed. The prevalence of appetite loss was 25.4% (95% confidence interval [CI], 21.5-29.4). High severity of depression was associated with a high probability of appetite loss (odds ratio, 1.2; 95%CI, 1.14-1.28; p = 0.00). Poor appetite is common 12 months after intensive care and is associated with the severity of depression.

Employment status and its associated factors for patients 12 months after intensive care: Secondary analysis of the SMAP-HoPe study.

BACKGROUND: Returning to work is a serious issue that affects patients who are discharged from the intensive care unit (ICU). This study aimed to clarify the employment status and the perceived household financial status of ICU patients 12 months following ICU discharge. Additionally, we evaluated whether there exists an association between depressive symptoms and subsequent unemployment status. METHODS: This study was a subgroup analysis of the published Survey of Multicenter Assessment with Postal questionnaire for Post-Intensive Care Syndrome for Home Living Patients (the SMAP-HoPe study) in Japan. Eligible patients were those who were employed before ICU admission, stayed in the ICU for at least three nights between October 2019 and July 2020, and lived at home for 12 months after discharge. We assessed the employment status, subjective cognitive functions, household financial status, Hospital Anxiety and Depression Scale, and EuroQOL-5 dimensions of physical function at 12 months following intensive care. RESULTS: This study included 328 patients, with a median age of 64 (interquartile range [IQR], 52-72) years. Of these, 79 (24%) were unemployed 12 months after ICU discharge. The number of patients who reported worsened financial status was significantly higher in the unemployed group (p<0.01) than in the employed group. Multivariable analysis showed that higher age (odds ratio [OR], 1.06; 95% confidence interval [CI], 1.03-1.08]) and greater severity of depressive symptoms (OR, 1.13 [95% CI, 1.05-1.23]) were independent factors for unemployment status at 12 months after ICU discharge. CONCLUSIONS: We found that 24.1% of our patients who had been employed prior to ICU admission were subsequently unemployed following ICU discharge and that depressive symptoms were associated with unemployment status. The government and the local municipalities should provide medical and financial support to such patients. Additionally, community and workplace support for such patients are warranted.

Prevalence of and risk factors for post-intensive care syndrome: Multicenter study of patients living at home after treatment in 12 Japanese intensive care units, SMAP-HoPe study.

Few studies have examined the epidemiology of post-intensive care syndrome in Japan. This study investigated the mental health and quality of life of patients living at home in Japan after intensive care unit (ICU) discharge. Additionally, we examined whether unplanned admission to the ICU was associated with more severe post-traumatic stress disorder (PTSD), anxiety, and depressive symptoms. An ambidirectional cohort study was conducted at 12 ICUs in Japan. Patients who stayed in the ICU for > 3 nights and were living at home for 1 year afterward were included. One year after ICU discharge, we retrospectively screened patients and performed a mail survey on a monthly basis, including the Impact of Event Scale-Revised (IER-S), the Hospital Anxiety Depression Scale (HADS), and the EuroQOL-5 Dimension (EQ-5D-L) questionnaires. Patients' characteristics, delirium and coma status, drugs used, and ICU and hospital length of stay were assessed from medical records. Descriptive statistics and multilevel linear regression modeling were used to examine our hypothesis. Among 7,030 discharged patients, 854 patients were surveyed by mail. Of these, 778 patients responded (response rate = 91.1%). The data from 754 patients were analyzed. The median IES-R score was 3 (interquartile range [IQR] = 1‒9), and the prevalence of suspected PTSD was 6.0%. The median HADS anxiety score was 4.00 (IQR = 1.17‒6.00), and the prevalence of anxiety was 16.6%. The median HADS depression score was 5 (IQR = 2‒8), and the prevalence of depression was 28.1%. EQ-5D-L scores were lower in our participants than in the sex- and age-matched Japanese population. Unplanned admission was an independent risk factor for more severe PTSD, anxiety, and depressive symptoms. Approximately one-third of patients in the general ICU population experienced mental health issues one year after ICU discharge. Unplanned admission was an independent predictor for more severe PTSD symptoms.

Flavonoids as substrates and inhibitors of myeloperoxidase: molecular actions of aglycone and metabolites.

Myeloperoxidase (MPO), secreted by activated neutrophils and macrophages at the site of inflammation, may be implicated in the oxidation of protein/lipoprotein during the development of cardiovascular diseases. Flavonoids have been suggested to act as antioxidative and anti-inflammatory agents in vivo; however, their molecular actions have not yet been fully understood. In this study, we examined the molecular basis of the inhibitory effects of dietary flavonoids, such as quercetin, and their metabolites on the catalytic reaction of MPO using a combination of biological assays and theoretical calculation studies. Immunohistochemical staining showed that a quercetin metabolite was colocalized with macrophages, MPO, and dityrosine, an MPO-derived oxidation product of tyrosine, in human atherosclerotic aorta. Quercetin and the plasma metabolites inhibited the formation of dityrosine catalyzed by the MPO enzyme and HL-60 cells in a dose-dependent manner. Spectrometric analysis indicated that quercetin might act as a cosubstrate of MPO resulting in the formation of the oxidized quercetin. Quantitative structure-activity relationship studies showed that the inhibitory actions of flavonoids strongly depended not only on radical scavenging activity but also on hydrophobicity (log P). The requirement of a set of hydroxyl groups at the 3, 5, and 4'-positions and C2-C3 double bond was suggested for the inhibitory effect. The binding of quercetin and the metabolites to a hydrophobic region at the entrance to the distal heme pocket of MPO was also proposed by a computer docking simulation. The current study provides the structure-activity relationships for flavonoids as the anti-inflammatory dietary constituents targeting the MPO-derived oxidative reactions in vivo.

[Experience with home visits for treatment of dysphagia (Clinical meaning of severity grading of dysphagia)].

It is more difficult to adequately manage dsyphagia patients at home than in the hospital. Based on the author's experience with home visits, grading the severity of dysphagia is advocated as helpful in reducing the difficulty. Both the clinical meaning and usefulness of severity grading are discussed. A diagnostic procedure designed by the author and ENT colleagues was applied to patients complaining of dysphagia at home. The procedure consists of three components, i.e., a questionnaire chart, endoscopic survey of the throat while swallowing and an assessment of the patient's general condition. Analysis of 24 cases revealed that the severity of dysphagia could be classified into four degrees, and that the the severity was helpful for determing the protocol of subsequent treatment. The part of the management of dysphagia at home in which the ENT physician can participate has been clarified.

Non-Canonical Role of IKKα in the Regulation of STAT1 Phosphorylation in Antiviral Signaling.

Non-self RNA is recognized by retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), inducing type I interferons (IFNs). Type I IFN promotes the expression of IFN-stimulated genes (ISGs), which requires the activation of signal transducer and activator of transcription-1 (STAT1). We previously reported that dsRNA induced STAT1 phosphorylation via a type I IFN-independent pathway in addition to the well-known type I IFN-dependent pathway. IκB kinase α (IKKα) is involved in antiviral signaling induced by dsRNA; however, its role is incompletely understood. Here, we explored the function of IKKα in RLR-mediated STAT1 phosphorylation. Silencing of IKKα markedly decreased the level of IFN-ß and STAT1 phosphorylation inHeH response to dsRNA. However, the inhibition of IKKα did not alter the RLR signaling-mediated dimerization of interferon responsive factor 3 (IRF3) or the nuclear translocation of nuclear factor-κB (NFκB). These results suggest a non-canonical role of IKKα in RLR signaling. Furthermore, phosphorylation of STAT1 was suppressed by IKKα knockdown in cells treated with a specific neutralizing antibody for the type I IFN receptor (IFNAR) and in IFNAR-deficient cells. Collectively, the dual regulation of STAT1 by IKKα in antiviral signaling suggests a role for IKKα in the fine-tuning of antiviral signaling in response to non-self RNA.

cis-3-Hexenol and trans-2-hexenal mixture prevents development of PTSD-like phenotype in rats.

Several green leaf volatiles have anxiolytic/antidepressant properties and attenuate adrenocortical stress response in rodents. However, it remains unknown whether a mixture of cis-3-hexenol and trans-2-hexenal so-called 'green odor (GO)' affects fear-associated post-traumatic stress disorder (PTSD)-like behavior. In the present study, fear memory of the initial conditioning stimulus was stably maintained by weekly presentation of conditioned tone. Examination of open field behavior, acoustic startle response, prepulse inhibition, and immobility in the forced swim test for 2 weeks after initial conditioning revealed that conditioned rats sustained anxiety, enhanced startle response, hypervigilance, depression-like behavior, and hypocortisolism, which is consistent with PTSD symptoms. Daily, not acute, GO presentation facilitated fear extinction and reduced PTSD-like behavioral and endocrinal responses. To further investigate the mechanism of effect of GO, we examined the effect of paroxetine (a selective serotonin reuptake inhibitor), p-chlorophenylalanine (PCPA, an irreversible serotonin synthesis inhibitor), alone or in combination of GO on PTSD-like phenotype. The alleviative effects of GO were masked by simultaneous paroxetine administration. PCPA-induced serotonin depletion abolished the effects of GO. Our results suggest that daily GO presentation facilitates fear extinction and prevents development of PTSD-like symptoms.

Spontaneous epileptic seizures in transgenic rats harboring a human ADNFLE missense mutation in the ß2-subunit of the nicotinic acetylcholine receptor.

We generated a transgenic rat strain with a missense mutation in V286L (V286L-TG), in the gene encoding the neuronal nicotinic acetylcholine receptor ß2 subunit (CHRNB2) found in patients with autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). To confirm that V286L-TG rats exhibit seizures similar to those observed in humans, gene expression patterns and behavioral phenotypes were analyzed. In situ hybridization using a V286L Chrnb2-selective probe indicated that the transgene was expressed at higher levels in the cortex, hippocampus, and cerebellum of V286L-TG than wild-type littermates (non-TG). Spontaneous epileptic seizures with ictal discharges in electroencephalograms were detected in 45% of V286L-TG rats and the frequency of seizures was 0.73 times a week. This seizure type is similar to "paroxysmal arousals" that are observed in human ADNFLE. V286L-TG rats displayed nicotine-induced abnormal motor activity including seizures in comparison to non-TGs. Response time following nicotine administration occurred faster in V286L-TG than in non-TG rats. V286L-TG rats demonstrated spontaneous epileptic seizures, which are similar to human ADNFLE, and also showed a higher sensitivity to nicotine administration. Thus, the V286L-TG rat model could be a valuable tool for developing novel mechanism-driven treatment strategies for epilepsy and provide a better understanding of ADNFLE.

Mitochondrial dysfunction leads to deconjugation of quercetin glucuronides in inflammatory macrophages.

Dietary flavonoids, such as quercetin, have long been recognized to protect blood vessels from atherogenic inflammation by yet unknown mechanisms. We have previously discovered the specific localization of quercetin-3-O-glucuronide (Q3GA), a phase II metabolite of quercetin, in macrophage cells in the human atherosclerotic lesions, but the biological significance is poorly understood. We have now demonstrated the molecular basis of the interaction between quercetin glucuronides and macrophages, leading to deconjugation of the glucuronides into the active aglycone. In vitro experiments showed that Q3GA was bound to the cell surface proteins of macrophages through anion binding and was readily deconjugated into the aglycone. It is of interest that the macrophage-mediated deconjugation of Q3GA was significantly enhanced upon inflammatory activation by lipopolysaccharide (LPS). Zymography and immunoblotting analysis revealed that ß-glucuronidase is the major enzyme responsible for the deglucuronidation, whereas the secretion rate was not affected after LPS treatment. We found that extracellular acidification, which is required for the activity of ß-glucuronidase, was significantly induced upon LPS treatment and was due to the increased lactate secretion associated with mitochondrial dysfunction. In addition, the ß-glucuronidase secretion, which is triggered by intracellular calcium ions, was also induced by mitochondria dysfunction characterized using antimycin-A (a mitochondrial inhibitor) and siRNA-knockdown of Atg7 (an essential gene for autophagy). The deconjugated aglycone, quercetin, acts as an anti-inflammatory agent in the stimulated macrophages by inhibiting the c-Jun N-terminal kinase activation, whereas Q3GA acts only in the presence of extracellular ß-glucuronidase activity. Finally, we demonstrated the deconjugation of quercetin glucuronides including the sulfoglucuronides in vivo in the spleen of mice challenged with LPS. These results showed that mitochondrial dysfunction plays a crucial role in the deconjugation of quercetin glucuronides in macrophages. Collectively, this study contributes to clarifying the mechanism responsible for the anti-inflammatory activity of dietary flavonoids within the inflammation sites.

A novel prophylactic effect of furosemide treatment on autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE).

The transgenic rat strain S284L-TG harbors the S284L mutant of the neuronal nicotinic acetylcholine receptor alpha4 subunit gene (CHRNA4), which is responsible for human autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). S284L-TG rats have epileptic seizure phenotypes during slow-wave sleep, similar to those in NFLE. We previously demonstrated that γ-aminobutyric acid (GABA)ergic action of these rats was suppressed before the onset of ADNFLE seizures, and that glutamate release in the epileptic focus lesion was increased at the onset of epilepsy. Here, mRNA analysis revealed that Cl(-)-accumulating Na-K-2Cl cotransporter 1 (NKCC1) levels were increased and Cl(-)-extruding K-Cl cotransporter 1 and 2 (KCC1 and KCC2) levels were decreased at the onset of ADNFLE seizures in S284L-TG rat frontal cortexes, which perturbed the GABAergic inhibitory system. The reversal potentials (EGABA) of GABAA receptor-mediated currents in cortical layer V pyramidal neurons of S284L-TG rats also changed their polarity from hyperpolarization to depolarization, and S284L-TG miniature excitatory postsynaptic currents (mEPSCs), but not miniature inhibitory postsynaptic currents (mIPSCs), significantly increased in both amplitude and frequency. Administration of 25mg/kg/day furosemide before, but not after, the onset of interictal discharges prevented idiopathic epileptic activity, reversed the depolarizing shift of EGABA and increased mEPSC amplitude to normal levels. These data indicate that early treatment with an agent that normalizes pathogenesis has a prophylactic effect on epilepsy. We propose a strategy for prophylactic medication against idiopathic epilepsy through the suppression of epileptogenesis and/or ictogenesis.

Macrophage as a target of quercetin glucuronides in human atherosclerotic arteries: implication in the anti-atherosclerotic mechanism of dietary flavonoids.

Epidemiological studies suggest that the consumption of flavonoid-rich diets decreases the risk of cardiovascular diseases. However, the target sites of flavonoids underlying the protective mechanism in vivo are not known. Quercetin represents antioxidative/anti-inflammatory flavonoids widely distributed in the human diet. In this study, we raised a novel monoclonal antibody 14A2 targeting the quercetin-3-glucuronide (Q3GA), a major antioxidative quercetin metabolite in human plasma, and found that the activated macrophage might be a potential target of dietary flavonoids in the aorta. Immunohistochemical studies with monoclonal antibody 14A2 demonstrated that the positive staining specifically accumulates in human atherosclerotic lesions, but not in the normal aorta, and that the intense staining was primarily associated with the macrophage-derived foam cells. In vitro experiments with murine macrophage cell lines showed that the Q3GA was significantly taken up and deconjugated into the much more active aglycone, a part of which was further converted to the methylated form, in the activated macrophages. In addition, the mRNA expression of the class A scavenger receptor and CD36, which play an important role for the formation of foam cells, was suppressed by the treatment of Q3GA. These results suggest that injured/inflamed arteries with activated macrophages are the potential targets of the metabolites of dietary quercetin. Our data provide a new insight into the bioavailability of dietary flavonoids and the mechanism for the prevention of cardiovascular diseases.