Protective role of cytosolic prion protein against virus infection in prion-infected cells.

Production of the amyloidogenic prion protein, PrPSc, which forms infectious protein aggregates, or prions, is a key pathogenic event in prion diseases. Functional prion-like protein aggregations, such as the mitochondrial adaptor protein MAVS and the inflammasome component protein ASC, have been identified to play a protective role in viral infections in mammalian cells. In this study, to investigate if PrPSc could play a functional role against external stimuli, we infected prion-infected cells with a neurotropic influenza A virus strain, IAV/WSN. We found that prion-infected cells were highly resistant to IAV/WSN infection. In these cells, NF-κB nuclear translocation was disturbed; therefore, mitochondrial superoxide dismutase (mtSOD) expression was suppressed, and mitochondrial reactive oxygen species (mtROS) was increased. The elevated mtROS subsequently activated NLRP3 inflammasomes, leading to the suppression of IAV/WSN-induced necroptosis. We also found that prion-infected cells accumulated a portion of PrP molecules in the cytosol, and that the N-terminal potential nuclear translocation signal of PrP impeded NF-κB nuclear translocation. These results suggest that PrPSc might play a functional role in protection against viral infections by stimulating the NLRP3 inflammasome-dependent antivirus mechanism through the cytosolic PrP-mediated disturbance of NF-κB nuclear translocation, which leads to suppression of mtSOD expression and consequently upregulation of the NLRP3 inflammasome activator mtROS. IMPORTANCE: Cytosolic PrP has been detected in prion-infected cells and suggested to be involved in the neurotoxicity of prions. Here, we also detected cytosolic PrP in prion-infected cells. We further found that the nuclear translocation of NF-κB was disturbed in prion-infected cells and that the N-terminal potential nuclear translocation signal of PrP expressed in the cytosol disturbed the nuclear translocation of NF-κB. Thus, the N-terminal nuclear translocation signal of cytosolic PrP might play a role in prion neurotoxicity. Prion-like protein aggregates in other protein misfolding disorders, including Alzheimer's disease were reported to play a protective role against various environmental stimuli. We here showed that prion-infected cells were partially resistant to IAV/WSN infection due to the cytosolic PrP-mediated disturbance of the nuclear translocation of NF-κB, which consequently activated NLRP3 inflammasomes after IAV/WSN infection. It is thus possible that prions could also play a protective role in viral infections.

Bakuchiol targets mitochondrial proteins, prohibitins and voltage-dependent anion channels: New insights into developing antiviral agents.

We previously reported that bakuchiol, a phenolic isoprenoid anticancer compound, and its analogs exert anti-influenza activity. However, the proteins targeted by bakuchiol remain unclear. Here, we investigated the chemical structures responsible for the anti-influenza activity of bakuchiol and found that all functional groups and C6 chirality of bakuchiol were required for its anti-influenza activity. Based on these results, we synthesized a molecular probe containing a biotin tag bound to the C1 position of bakuchiol. With this probe, we performed a pulldown assay for Madin-Darby canine kidney cell lysates and purified the specific bakuchiol-binding proteins with SDS-PAGE. Using nanoLC-MS/MS analysis, we identified prohibitin (PHB) 2, voltage-dependent anion channel (VDAC) 1, and VDAC2 as binding proteins of bakuchiol. We confirmed the binding of bakuchiol to PHB1, PHB2, and VDAC2 in vitro using Western blot analysis. Immunofluorescence analysis showed that bakuchiol was bound to PHBs and VDAC2 in cells and colocalized in the mitochondria. The knockdown of PHBs or VDAC2 by transfection with specific siRNAs, along with bakuchiol cotreatment, led to significantly reduced influenza nucleoprotein expression levels and viral titers in the conditioned medium of virus-infected Madin-Darby canine kidney cells, compared to the levels observed with transfection or treatment alone. These findings indicate that reducing PHBs or VDAC2 protein, combined with bakuchiol treatment, additively suppressed the growth of influenza virus. Our findings indicate that bakuchiol exerts anti-influenza activity via a novel mechanism involving these mitochondrial proteins, providing new insight for developing anti-influenza agents.

Clinical Utility of SARS-CoV-2 Antibody Titer Multiplied by Binding Avidity of Receptor-Binding Domain (RBD) in Monitoring Protective Immunity and Clinical Severity.

Conventional serum antibody titer, which expresses antibody level, does not provide antigen binding avidity of the variable region of the antibody, which is essential for the defense response to infection. Here, we quantified anti-SARS-CoV-2 antibody binding avidity to the receptor-binding domain (RBD) by competitive binding-inhibition activity (IC50) between SARS-CoV-2 S1 antigen immobilized on the DCP microarray and various RBD doses added to serum and expressed as 1/IC50 nM. The binding avidity analyzed under equilibrium conditions of antigen-antibody binding reaction is different from the avidity index measured with the chaotropic agent, such as urea, under nonequilibrium and short-time conditions. Quantitative determination of the infection-protection potential of antibodies was assessed by ABAT (antigen binding avidity antibody titer), which was calculated by the quantity (level) × quality (binding avidity) of antibodies. The binding avidity correlated strongly (r = 0.811) with cell-based virus-neutralizing activity. Maturation of the protective antibody induced by repeated vaccinations or SARS-CoV-2 infection was classified into three categories of ABAT, such as an initial, low, and high ABAT. Antibody maturity correlated with the clinical severity of COVID-19. Once a mature high binding avidity was achieved, it was maintained for at least 6-8 months regardless of the subsequent change in the antibody levels.

Effect of Maternal Egg Intake During the Early Neonatal Period and Risk of Infant Egg Allergy at 12 Months Among Breastfeeding Mothers: A Randomized Clinical Trial.

Importance: Egg introduction in infants at age 4 to 6 months is associated with a lower risk of immunoglobulin E-mediated egg allergy (EA). However, whether their risk of EA at age 12 months is affected by maternal intake of eggs at birth is unknown. Objective: To determine the effect of maternal egg intake during the early neonatal period (0-5 days) on the development of EA in breastfed infants at age 12 months. Design, Setting, and Participants: This multicenter, single-blind (outcome data evaluators), randomized clinical trial was conducted from December 18, 2017, to May 31, 2021, at 10 medical facilities in Japan. Newborns with at least 1 of 2 parents having an allergic disease were included. Neonates whose mothers had EA or were unable to consume breast milk after the age of 2 days were excluded. Data were analyzed on an intention-to-treat basis. Interventions: Newborns were randomized (1:1) to a maternal egg consumption (MEC) group, wherein the mothers consumed 1 whole egg per day during the first 5 days of the neonate's life, and a maternal egg elimination (MEE) group, wherein the mothers eliminated eggs from their diet during the same period. Main Outcomes and Measures: The primary outcome was EA at age 12 months. Egg allergy was defined as sensitization to egg white or ovomucoid plus a positive test result in an oral food challenge or an episode of obvious immediate symptoms after egg ingestion. Results: Of the 380 newborns included (198 [52.1%] female), 367 (MEC: n = 183; MEE: n = 184) were followed up for 12 months. On days 3 and 4 after delivery, the proportions of neonates with ovalbumin and ovomucoid detection in breast milk were higher in the MEC group than in the MEE group (ovalbumin: 10.7% vs 2.0%; risk ratio [RR], 5.23; 95% CI, 1.56-17.56; ovomucoid: 11.3% vs 2.0%; RR, 5.55; 95% CI, 1.66-18.55). At age 12 months, the MEC and MEE groups did not differ significantly in EA (9.3% vs 7.6%; RR, 1.22; 95% CI, 0.62-2.40) or sensitization to egg white (62.8% vs 58.7%; RR, 1.07; 95% CI, 0.91-1.26). No adverse effects were reported. Conclusions and Relevance: In this randomized clinical trial, EA development and sensitization to eggs were unaffected by MEC during the early neonatal period. Trial Registration: UMIN Clinical Trials Registry: UMIN000027593.

The Combination of Binding Avidity of Ovomucoid-Specific IgE Antibody and Specific IgG4 Antibody Can Predict Positive Outcomes of Oral Food Challenges during Stepwise Slow Oral Immunotherapy in Children with Hen's Egg Allergy.

To increase the prediction accuracy of positive oral food challenge (OFC) outcomes during stepwise slow oral immunotherapy (SS-OIT) in children with a hen's egg (HE) allergy, we evaluated the predictive value of the combination of antigen-specific IgE (sIgE) with antigen binding avidity and sIgG4 values. Sixty-three children with HE allergy undergoing SS-OIT were subjected to repeated OFCs with HE. We measured the ovomucoid (OVM)-sIgE by ImmunoCAP or densely carboxylated protein (DCP) microarray, sIgG4 by DCP microarray, and the binding avidity of OVM-sIgE defined as the level of 1/IC50 (nM) measured by competitive binding inhibition assays. The OFC was positive in 37 (59%) patients undergoing SS-OIT. Significant differences in DCP-OVM-sIgE, CAP-OVM-sIgE, I/IC50, DCP-OVM-sIgG4, the multiplication products of DCP-OVM-sIgE, and the binding avidity of DCP-OVM-sIgE (DCP-OVM-sIgE/IC50) and DCP-OVM-sIgE/sIgG4 were compared between the negative and positive groups (p < 0.01). Among them, the variable with the greatest area under the receiver operating characteristic curve was DCP-OVM-sIgE/IC50 (0.84), followed by DCP-OVM-sIgE/sIgG4 (0.81). DCP-OVM-sIgE/IC50 and DCP-OVM-sIgE/sIgG4 are potentially useful markers for the prediction of positive OFCs during HE-SS-OIT and may allow proper evaluation of the current allergic status in the healing process during HE-SS-OIT.

Anti-influenza Activity of Povidone-Iodine-Integrated Materials.

Personal protective equipment (PPE), including medical masks, should be worn for preventing the transmission of respiratory pathogens via infective droplets and aerosols. In medical masks, the key layer is the filter layer, and the melt-blown nonwoven fabric (NWF) is the most used fabric. However, the NWF filter layer cannot kill or inactivate the pathogens spread via droplets and aerosols. Povidone-iodine (PVP-I) has been used as an antiseptic solution given its potent broad-spectrum activity against pathogens. To develop PPE (e.g., medical masks) with anti-pathogenic activity, we integrated PVP-I into nylon-66 NWF. We then evaluated its antiviral activity against influenza A viruses by examining the viability of Madin-Darby canine kidney (MDCK) cells after inoculation with the virus strains exposed to the PVP-I-integrated nylon-66 NWF. The PVP-I nylon-66 NWF protected the MDCK cells from viral infection in a PVP-I concentration-dependent manner. Subsequently, we found to integrate PVP-I into nylon-66 and polyurethane materials among various materials. These PVP-I materials were also effective against influenza virus infection, and treatment with PVP-I nylon-66 NWF showed the highest cell survival among all the tested materials. PVP-I showed anti-influenza A virus activity when used in conjunction with PPE materials. Moreover, nylon-66 NWF integrated with PVP-I was found to be the best material to ensure anti-influenza activity. Therefore, PVP-I-integrated masks could have the potential to inhibit respiratory virus infection. Our results provide new information for developing multi-functional PPEs with anti-viral activity by integrating them with PVP-I to prevent the potential transmission of respiratory viruses.

Induction of systemic, mucosal, and cellular immunity against SARS-CoV-2 in mice vaccinated by trans-airway with a S1 protein combined with a pulmonary surfactant-derived adjuvant SF-10.

Background: There is a need for vaccines that can induce effective systemic, respiratory mucosal, and cellular immunity to control the COVID-19 pandemic. We reported previously that a synthetic mucosal adjuvant SF-10 derived from human pulmonary surfactant works as an efficient antigen delivery vehicle to antigen presenting cells in the respiratory and gastrointestinal tracts and promotes induction of influenza virus antigen-specific serum IgG, mucosal IgA, and cellular immunity. Methods: The aim of the present study was to determine the effectiveness of a new administration route of trans-airway (TA) vaccine comprising recombinant SARS-CoV-2 spike protein 1 (S1) combined with SF-10 (S1-SF-10 vaccine) on systemic, local, and cellular immunity in mice, compared with intramuscular injection (IM) of S1 with a potent adjuvant AddaS03™ (S1-AddaS03™ vaccine). Results: S1-SF-10-TA vaccine induced S1-specific IgG and IgA in serum and lung mucosae. These IgG and IgA induced by S1-SF-10-TA showed significant protective immunity in a receptor binding inhibition test of S1 and angiotensin converting enzyme 2, a receptor of SARS-CoV-2, which were more potent and faster achievement than S1-AddaS03™-IM. Enzyme-linked immunospot assay showed high numbers of S1-specific IgA and IgG secreting cells (ASCs) and S1-responsive IFN-γ, IL-4, IL-17A cytokine secreting cells (CSCs) in the spleen and lungs. S1-AddaS03™-IM induced IgG ASCs and IL-4 CSCs in spleen higher than S1-SF-10-TA, but the numbers of ASCs and CSCs in lungs were low and hardly detected. Conclusions: Based on the need for effective systemic, respiratory, and cellular immunity, the S1-SF-10-TA vaccine seems promising mucosal vaccine against respiratory infection of SARS-CoV-2.

A Murine Model of Food Allergy by Epicutaneous Adjuvant-Free Allergen Sensitization Followed by Oral Allergen Challenge Combined with Aspirin for Enhanced Detection of Hypersensitivity Manifestations and Immunotherapy Monitoring.

Food allergy is one of the major existing health problems, but no effective treatment is available. In the current work, a murine model that closely mimics pathogenesis of human food allergy and its quantifiable diagnostic parameter design, even for mild hypersensitivity reactions, were established. BALB/c mice were epicutaneously sensitized with 1 mg chicken egg ovomucoid (OVM) or cow's milk casein, free of adjuvants, five times a week for two consecutive weeks. Eleven days later, allergen-specific IgG1 and IgE in serum were measured by ELISA. On day 25, 20 mg OVM or 12 mg α-casein was administered orally, and allergic reactions such as the fall in rectal temperature, symptom scores during 90-120 min, serum mast cell protease-1 and cytokine levels were monitored. The detection of mild allergic reactions due to adjuvant-free allergen sensitization and oral allergen challenge routes was amplified by the combination of oral allergen and aspirin administration simultaneously or aspirin administration within 15-30 min before an allergen challenge. Quantification of the maximum symptom score and the frequency of symptoms during the monitoring period improved evaluation accuracy of food allergy signals. Based on these results, efficacy of casein oral immunotherapy for cow's milk allergies, which are generally difficult to detect, was monitored adequately.

A Potent PDK4 Inhibitor for Treatment of Heart Failure with Reduced Ejection Fraction.

Heart failure with reduced ejection fraction (HFrEF) is characterized not only by reduced left ventricular ejection fraction (EF) but is also combined with symptoms such as dyspnea, fatigue, and edema. Several pharmacological interventions have been established. However, a treatment targeting a novel pathophysiological mechanism is still needed. Evidence indicating that inhibition of pyruvate dehydrogenase kinase 4 (PDK4) may be cardioprotective has been accumulating. Thus, we focused on vitamin K3 and used its framework as a new PDK4 inhibitor skeleton to synthesize new PDK4 inhibitors that show higher activity than the existing PDK4 inhibitor, dichloroacetic acid, and tested their cardioprotective effects on a mouse heart failure model. Among these inhibitors, PDK4 inhibitor 8 improved EF the most, even though it did not reverse cardiac fibrosis or wall thickness. This novel, potent PDK4 inhibitor may improve EF of failing hearts by regulating bioenergetics via activation of the tricarboxylic acid cycle.

Electrochemical enzyme-based blood ATP and lactate sensor for a rapid and straightforward evaluation of illness severity.

This study aimed to develop an electrochemical system for measuring blood ATP and lactate levels in a single format. The ratio of lactate to ATP levels was previously reported to provide an alternative illness severity score. Although severity evaluation is crucial to treat patients with acute disease admitted to intensive care units, no sensors are currently available to simply and rapidly measure ATP and lactate levels using the same detection method. Therefore, we constructed an integrated sensing system for ATP and lactate using enzymatic reactions and two sets of electrodes integrated into a chip connected to a single potentiostat operated by a microcontroller. The enzymatic system involves adenylate kinase, pyruvate kinase, and pyruvate oxidase for ATP, and lactate oxidase for lactate, both of which produce hydrogen peroxide. Multiplex enzyme-based reactions were designed to minimize the corresponding operations significantly without enzyme immobilization onto the electrodes. The system was robust in the presence of potentially interfering blood components, such as ascorbate, pyruvate, ADP, urate, and potassium ions. The ATP and lactate levels in the blood were successfully measured using the new sensor with good recoveries. The analytical results of blood samples obtained using our sensor were in good agreement with those using conventional methods. Integrating electrode-based analysis and a microcontroller-based system saved further operations, enabling the straightforward measurement of ATP and lactate levels within 5 min. The proposed sensor may serve as a useful tool in the management of serious infectious diseases.

Neurotropic influenza A virus infection causes prion protein misfolding into infectious prions in neuroblastoma cells.

Misfolding of the cellular prion protein, PrPC, into the amyloidogenic isoform, PrPSc, which forms infectious protein aggregates, the so-called prions, is a key pathogenic event in prion diseases. No pathogens other than prions have been identified to induce misfolding of PrPC into PrPSc and propagate infectious prions in infected cells. Here, we found that infection with a neurotropic influenza A virus strain (IAV/WSN) caused misfolding of PrPC into PrPSc and generated infectious prions in mouse neuroblastoma cells through a hit-and-run mechanism. The structural and biochemical characteristics of IAV/WSN-induced PrPSc were different from those of RML and 22L laboratory prions-evoked PrPSc, and the pathogenicity of IAV/WSN-induced prions were also different from that of RML and 22L prions, suggesting IAV/WSN-specific formation of PrPSc and infectious prions. Our current results may open a new avenue for the role of viral infection in misfolding of PrPC into PrPSc and formation of infectious prions.

Organic synthesis and anti-influenza A virus activity of cyclobakuchiols A, B, C, and D.

Novel antiviral agents for influenza, which poses a substantial threat to humans, are required. Cyclobakuchiols A and B have been isolated from Psoralea glandulosa, and cyclobakuchiol C has been isolated from P. corylifolia. The structural differences between cyclobakuchiol A and C arise due to the oxidation state of isopropyl group, and these compounds can be derived from (+)-(S)-bakuchiol, a phenolic isoprenoid compound present in P. corylifolia seeds. We previously reported that bakuchiol induces enantiospecific anti-influenza A virus activity involving nuclear factor erythroid 2-related factor 2 (Nrf2) activation. However, it remains unclear whether cyclobakuchiols A-C induce anti-influenza A virus activity. In this study, cyclobakuchiols A, B, and C along with cyclobakuchiol D, a new artificial compound derived from cyclobakuchiol B, were synthesized and examined for their anti-influenza A virus activities using Madin-Darby canine kidney cells. As a result, cyclobakuchiols A-D were found to inhibit influenza A viral infection, growth, and the reduction of expression of viral mRNAs and proteins in influenza A virus-infected cells. Additionally, these compounds markedly reduced the mRNA expression of the host cell influenza A virus-induced immune response genes, interferon-ß and myxovirus-resistant protein 1. In addition, cyclobakuchiols A-D upregulated the mRNA levels of NAD(P)H quinone oxidoreductase 1, an Nrf2-induced gene, in influenza A virus-infected cells. Notably, cyclobakuchiols A, B, and C, but not D, induced the Nrf2 activation pathway. These findings demonstrate that cyclobakuchiols have anti-influenza viral activity involving host cell oxidative stress response. In addition, our results suggest that the suitably spatial configuration between oxidized isopropyl group and phenol moiety in the structure of cyclobakuchiols is required for their effect.

A novel aqueous extract from rice fermented with Aspergillus oryzae and Saccharomyces cerevisiae possesses an anti-influenza A virus activity.

Human influenza virus infections occur annually worldwide and are associated with high morbidity and mortality. Hence, development of novel anti-influenza drugs is urgently required. Rice Power® extract developed by the Yushin Brewer Co. Ltd. is a novel aqueous extract of rice obtained via saccharization and fermentation with various microorganisms, such as Aspergillus oryzae, yeast [such as Saccharomyces cerevisiae], and lactic acid bacteria, possessing various biological and pharmacological properties. In our previous experimental screening with thirty types of Rice Power® extracts, we observed that the 30th Rice Power® (Y30) extract promoted the survival of influenza A virus-infected Madin-Darby canine kidney (MDCK) cells. Therefore, to identify compounds for the development of novel anti-influenza drugs, we aimed to investigate whether the Y30 extract exhibits anti-influenza A virus activity. In the present study, we demonstrated that the Y30 extract strongly promoted the survival of influenza A H1N1 Puerto Rico 8/34 (A/PR/8/34), California 7/09, or H3N2 Aichi 2/68 (A/Aichi/2/68) viruses-infected MDCK cells and inhibited A/PR/8/34 or A/Aichi/2/68 viruses infection and growth in the co-treatment and pre-infection experiments. The pre-treatment of Y30 extract on MDCK cells did not induce anti-influenza activity in the cell. The Y30 extract did not significantly affect influenza A virus hemagglutination, and neuraminidase and RNA-dependent RNA polymerase activities. Interestingly, the electron microscopy experiment revealed that the Y30 extract disrupts the integrity of influenza A virus particles by permeabilizing the viral membrane envelope, suggesting that Y30 extract has a direct virucidal effect against influenza A virus. Furthermore, we observed that compared to the ethyl acetate (EtOAc) extract, the water extract of Y30 extract considerably promoted the survival of cells infected with A/PR/8/34 virus. These results indicated that more anti-influenza components were present in the water extract of Y30 extract than in the EtOAc extract. Our results highlight the potential of a rice extract fermented with A. oryzae and S. cerevisiae as an anti-influenza medicine and a drug source for the development of anti-influenza compounds.

Prion protein signaling induces M2 macrophage polarization and protects from lethal influenza infection in mice.

The cellular prion protein, PrPC, is a glycosylphosphatidylinositol anchored-membrane glycoprotein expressed most abundantly in neuronal and to a lesser extent in non-neuronal cells. Its conformational conversion into the amyloidogenic isoform in neurons is a key pathogenic event in prion diseases, including Creutzfeldt-Jakob disease in humans and scrapie and bovine spongiform encephalopathy in animals. However, the normal functions of PrPC remain largely unknown, particularly in non-neuronal cells. Here we show that stimulation of PrPC with anti-PrP monoclonal antibodies (mAbs) protected mice from lethal infection with influenza A viruses (IAVs), with abundant accumulation of anti-inflammatory M2 macrophages with activated Src family kinases (SFKs) in infected lungs. A SFK inhibitor dasatinib inhibited M2 macrophage accumulation in IAV-infected lungs after treatment with anti-PrP mAbs and abolished the anti-PrP mAb-induced protective activity against lethal influenza infection in mice. We also show that stimulation of PrPC with anti-PrP mAbs induced M2 polarization in peritoneal macrophages through SFK activation in vitro and in vivo. These results indicate that PrPC could activate SFK in macrophages and induce macrophage polarization to an anti-inflammatory M2 phenotype after stimulation with anti-PrP mAbs, thereby eliciting protective activity against lethal infection with IAVs in mice after treatment with anti-PrP mAbs. These results also highlight PrPC as a novel therapeutic target for IAV infection.

A Double-Blind, Randomized, Placebo-Controlled Trial of Heat-Killed Pediococcus acidilactici K15 for Prevention of Respiratory Tract Infections among Preschool Children.

Although some probiotic bacteria have been reported to prevent infections in children, there are few well-designed double-blind studies. Here we evaluated the effects of a probiotic strain of lactic acid bacteria (LAB), Pediococcus acidilactici K15, on viral respiratory tract infections in preschool children. A four-month, randomized, double-blind, placebo-controlled study was performed in 172 healthy children aged 3 to 6 years. Subjects were administered dextrin alone or dextrin including heat-killed K15 (5 × 1010 bacteria). The number of febrile days was the primary outcome. The number of absent days from preschools and the influenza incidence were secondary outcomes. Secretory IgA (sIgA) concentrations in saliva were measured as an exploratory outcome. The primary and secondary outcomes were not significantly different between both groups. Analyses in children with little intake of fermented foods including LAB showed that the duration of a fever significantly decreased by K15 intake. The salivary sIgA level in the K15 group was maintained significantly higher than it was in the placebo group. The effects of K15 on preventing viral respiratory tract infections were not observed without the restriction of fermented foods intake. However, K15 supported anti-infectious immune systems in children who took less fermented foods and the maintenance of salivary sIgA levels in all subjects.

Role of host trypsin-type serine proteases and influenza virus-cytokine-trypsin cycle in influenza viral pathogenesis. Pathogenesis-based therapeutic options.

Influenza A virus (IAV) is one of the most common infectious pathogen and associated with significant morbidity and mortality. Although processing the IAV hemagglutinin (HA) envelope glycoprotein precursor is a pre-requisite for viral membrane fusion activity, viral entry and transmission, HA-processing protease is not encoded in the IAV genome and thus the cellular trypsin-type serine HA-processing proteases determine viral infectious tropism and viral pathogenicity. The initial process of IAV infection of the airway is followed by marked upregulation of ectopic trypsin in various organs and endothelial cells through the induction of various proinflammatory cytokines, and this process has been termed the "influenza virus-cytokine-trypsin" cycle. In the advanced stage of IAV infection, the cytokine storm induces disorders of glucose and lipid metabolism and the "metabolic disorders-cytokine" cycle is then linked with the "influenza virus-cytokine-trypsin" cycle, to advance the pathogenic process into energy crisis and multiple organ failure. Application of protease inhibitors and treatment of metabolic disorders that break these cycles and their interconnection is therefore a promising therapeutic approach against influenza. This review discusses IAV pathogenicity on trypsin type serine HA-processing proteases, cytokines, metabolites and therapeutic options.

Antitumor activity of potent pyruvate dehydrogenase kinase 4 inhibitors from plants in pancreatic cancer.

Phosphorylation of pyruvate dehydrogenase by pyruvate dehydrogenase kinase 4 (PDK4) 4 inhibits its ability to induce a glycolytic shift. PDK4 expression is frequently upregulated in various cancer tissues, with its elevation being critical for the induction of the Warburg effect. PDK4 is an attractive target for cancer therapy given its effect on shifting glucose metabolism. Previous research has highlighted the necessity of identifying a potent compound to suppress PDK4 activity at the submicromolar concentrations. Here we identified natural diterpene quinones (KIS compounds) that inhibit PDK4 at low micromolar concentrations. KIS37 (cryptotanshinone) inhibited anchorage-independent growth in three-dimensional spheroid and soft agar colony formation assays of KRAS-activated human pancreatic (MIAPaCa-2 and Panc-1) and colorectal (DLD-1 and HCT116) cancer cell lines. KIS37 also suppressed KRAS protein expression in such cell lines. Furthermore, KIS37 suppressed phosphorylation of Rb protein and cyclin D1 protein expression via the PI3K-Akt-mTOR signaling pathway under nonadherent culture conditions and suppressed the expression of cancer stem cell markers CD44, EpCAM, and ALDH1A1 in MIAPaCa-2 cells. KIS37 also suppressed pancreatic cancer cell growth in both subcutaneous xenograft and orthotopic pancreatic tumor models in nude mice at 40 mg/kg (intraperitoneal dose) without any evident toxicity. Reduced ALDH1A1 expression was observed in KIS37-treated pancreatic tumors, suggesting that cancer cell stemness was also suppressed in the orthotopic tumor model. The aforementioned results indicate that KIS37 administration is a novel therapeutic strategy for targeting PDK4 in KRAS-activated intractable human pancreatic cancer.