Negative Cross-Talk between TLR2/4-Independent AMPKα1 and TLR2/4-Dependent JNK Regulates S. pneumoniae-Induced Mucosal Innate Immune Response.

Streptococcus pneumoniae is major cause of otitis media (OM) and life-threatening pneumonia. Overproduction of mucin, the major component of mucus, plays a critical role in the pathogenesis of both OM and pneumonia. However, the molecular mechanisms underlying the tight regulation of mucin upregulation in the mucosal epithelium by S. pneumoniae infection remain largely unknown. In this study, we show that S. pneumoniae pneumolysin (PLY) activates AMP-activated protein kinase α1 (AMPKα1), the master regulator of energy homeostasis, which is required for S. pneumoniae-induced mucin MUC5AC upregulation in vitro and in vivo. Moreover, we found that PLY activates AMPKα1 via cholesterol-dependent membrane binding of PLY and subsequent activation of the Ca2+- Ca2+/calmodulin-dependent kinase kinase ß (CaMKKß) and Cdc42-mixed-lineage protein kinase 3 (MLK3) signaling axis in a TLR2/4-independent manner. AMPKα1 positively regulates PLY-induced MUC5AC expression via negative cross-talk with TLR2/4-dependent activation of MAPK JNK, the negative regulator of MUC5AC expression. Moreover, pharmacological inhibition of AMPKα1 suppressed MUC5AC induction in the S. pneumoniae-induced OM mouse model, thereby demonstrating its therapeutic potential in suppressing mucus overproduction in OM. Taken together, our data unveil a novel mechanism by which negative cross-talk between TLR2/4-independent activation of AMPKα1 and TLR2/4-dependent activation of JNK tightly regulates the S. pneumoniae PLY-induced host mucosal innate immune response.

Assessment of the probability of microbial contamination for sample return from Martian moons I: Departure of microbes from Martian surface.

Potential microbial contamination of Martian moons, Phobos and Deimos, which can be brought about by transportation of Mars ejecta produced by meteoroid impacts on the Martian surface, has been comprehensively assessed in a statistical approach, based on the most probable history of recent major gigantic meteoroid collisions on the Martian surface. This article is the first part of our study to assess potential microbial density in Mars ejecta departing from the Martian atmosphere, as a source of the second part (Kurosawa et al., 2019) where statistical analysis of microbial contamination probability is conducted. Potential microbial density on the Martian surface as the source of microorganisms was estimated by analogy to the terrestrial areas having the similar arid and cold environments, from which a probabilistic function was deduced as the asymptotic limit. Microbial survival rate during hypervelocity meteoroid collisions was estimated by numerical analysis of impact phenomena with and without taking internal friction and plastic deformation of the colliding meteoroid and the target ground into consideration. Trajectory calculations of departing ejecta through the Martian atmosphere were conducted with taking account of aerodynamic deceleration and heating by the aid of computational fluid dynamic analysis. It is found that Mars ejecta smaller than 0.03 m in diameter hardly reach the Phobos orbit due to aerodynamic deceleration, or mostly sterilized due to significant aerodynamic heating even though they can reach the Phobos orbit and beyond. Finally, the baseline dataset of microbial density in Mars ejecta departing for Martian moons has been presented for the second part of our study.

Assessment of the probability of microbial contamination for sample return from Martian moons II: The fate of microbes on Martian moons.

This paper presents a case study of microbe transportation in the Mars-satellites system. We examined the spatial distribution of potential impact-transported microbes on the Martian moons using impact physics by following a companion study (Fujita et al., in this issue). We used sterilization data from the precede studies (Patel et al., 2018; Summers, 2017). We considered that the microbes came mainly from the Zunil crater on Mars, which was formed during 1.0-0.1 Ma. We found that 70-80% of the microbes are likely to be dispersed all over the moon surface and are rapidly sterilized due to solar and galactic cosmic radiation except for those microbes within a thick ejecta deposit produced by natural meteoroids. The other 20-30% might be shielded from radiation by thick regolith layers that formed at collapsed layers in craters produced by Mars rock impacts. The total number of potentially surviving microbes at the thick ejecta deposits is estimated to be 3-4 orders of magnitude lower than at the Mars rock craters. The microbe concentration is irregular in the horizontal direction due to Mars rock bombardment and is largely depth-dependent due to the radiation sterilization. The surviving fraction of transported microbes would be only ∼1 ppm on Phobos and ∼100 ppm on Deimos, suggesting that the transport processes and radiation severely affect microbe survival. The microbe sampling probability from the Martian moons was also investigatesd. We suggest that sample return missions from the Martian moons are classified into Unrestricted Earth-Return missions for 30 g samples and 10 cm depth sampling, even in our conservative scenario. We also conducted a full statistical analysis pertaining to sampling the regolith of Phobos to include the effects of uncertainties in input parameters on the sampling probability. The most likely probability of microbial contamination for return samples is estimated to be two orders of magnitude lower than the 10-6 criterion defined by the planetary protection policy of the Committee on Space Research (COSPAR).

Spatiotemporal dynamics and early detection of thermoacoustic combustion instability in a model rocket combustor.

We numerically study the spatiotemporal dynamics and early detection of thermoacoustic combustion instability in a model rocket combustor using the theories of complex networks and synchronization. The turbulence network, which consists of nodes and vertexes in weighted networks between vortices, can characterize the complex spatiotemporal structure of a flow field during thermoacoustic combustion instability. The transfer entropy allows us to identify the driving region of thermoacoustic combustion instability. In addition to the order parameter, a phase parameter newly proposed in this study is useful for capturing the precursor of thermoacoustic combustion instability.

Early-Life Microbiota Exposure Restricts Myeloid-Derived Suppressor Cell-Driven Colonic Tumorigenesis.

Gut microbiota and their metabolites are instrumental in regulating homeostasis at intestinal and extraintestinal sites. However, the complex effects of prenatal and early postnatal microbial exposure on adult health and disease outcomes remain incompletely understood. Here, we showed that mice raised under germ-free conditions until weaning and then transferred to specific pathogen-free (SPF) conditions harbored altered microbiota composition, augmented inflammatory cytokine and chemokine expression, and were hyper-susceptible to colitis-associated tumorigenesis later in adulthood. Increased number and size of colon tumors and intestinal epithelial cell proliferation in recolonized germ-free mice were associated with augmented intratumoral CXCL1, CXCL2, and CXCL5 expression and granulocytic myeloid-derived suppressor cell (G-MDSC) accumulation. Consistent with these findings, CXCR2 neutralization in recolonized germ-free mice completely reversed the exacerbated susceptibility to colitis-associated tumorigenesis. Collectively, our findings highlight a crucial role for early-life microbial exposure in establishing intestinal homeostasis that restrains colon cancer in adulthood.

Spatiotemporal dynamics of turbulent coaxial jet analyzed by symbolic information-theory quantifiers and complex-network approach.

We numerically study the spatiotemporal dynamics of a turbulent coaxial jet in a model rocket engine combustor from the viewpoints of symbolic information-theory quantifiers and complex networks. The dynamic behavior of flow velocity undergoes a significant transition from a stochastic to chaotic state as the turbulent jet moves downstream. The small-world nature exists in the near field forming a stochastic state, whereas it disappears by the formation of a chaotic state in the far field. The dynamic behavior of hydrogen and oxygen concentrations in the far field also represents deterministic chaos. The simultaneous dynamic behavior with chaotic mixing forms the phase-synchronization state.

Taurine Inhibits TRPV-Dependent Activity to Overcome Oxidative Stress in Caenorhabditis elegans.

Taurine has important physiological roles as well as a wide range of pharmacological effects. Studies have suggested that taurine ameliorates diabetes, hypertension, oxidative stress, and inflammatory diseases. However, its mechanisms of action are still unclear. It has been reported that N-acyl taurine activates transient receptor potential vanilloid-1 (TRPV1), which is related to the pathogenesis of many inflammatory diseases. In this study, we hypothesized that taurine has a regulatory effect on TRPV1 activation via N-acyl taurine. To evaluate this hypothesis, we assessed the calcium influx activated by a TRPV1 agonist in human keratinocyte (HaCaT) cells and paraquat-induced oxidative stress in Caenorhabditis elegans. Our results indicate that taurine inhibits TRPV-dependent activity to overcome oxidative stress in cultured cell lines and in C. elegans.

Heterosubtypic influenza protection elicited by double-layered polypeptide nanoparticles in mice.

Influenza is a persistent threat to public health. Here we report that double-layered peptide nanoparticles induced robust specific immunity and protected mice against heterosubtypic influenza A virus challenges. We fabricated the nanoparticles by desolvating a composite peptide of tandem copies of nucleoprotein epitopes into nanoparticles as cores and cross-linking another composite peptide of four tandem copies of influenza matrix protein 2 ectodomain epitopes to the core surfaces as a coating. Delivering the nanoparticles via dissolvable microneedle patch-based skin vaccination further enhanced the induced immunity. These peptide-only, layered nanoparticles demonstrated a strong antigen depot effect and migrated into spleens and draining (inguinal) lymph nodes for an extended period compared with soluble antigens. This increased antigen-presentation time correlated with the stronger immune responses in the nanoparticle-immunized group. The protection conferred by nanoparticle immunization was transferable by passive immune serum transfusion and depended partially on a functional IgG receptor FcγRIV. Using a conditional cell depletion, we found that CD8+ T cells were involved in the protection. The immunological potency and stability of the layered peptide nanoparticles indicate applications for other peptide-based vaccines and peptide drug delivery.

A Split-Luciferase-Based Trimer Formation Assay as a High-throughput Screening Platform for Therapeutics in Alport Syndrome.

Alport syndrome is a hereditary glomerular disease caused by mutation in type IV collagen α3-α5 chains (α3-α5(IV)), which disrupts trimerization, leading to glomerular basement membrane degeneration. Correcting the trimerization of α3/α4/α5 chain is a feasible therapeutic approach, but is hindered by lack of information on the regulation of intracellular α(IV) chain and the absence of high-throughput screening (HTS) platforms to assess α345(IV) trimer formation. Here, we developed sets of split NanoLuc-fusion α345(IV) proteins to monitor α345(IV) trimerization of wild-type and clinically associated mutant α5(IV). The α345(IV) trimer assay, which satisfied the acceptance criteria for HTS, enabled the characterization of intracellular- and secretion-dependent defects of mutant α5(IV). Small interfering RNA-based and chemical screening targeting the ER identified several chemical chaperones that have potential to promote α345(IV) trimer formation. This split luciferase-based trimer formation assay is a functional HTS platform that realizes the feasibility of targeting α345(IV) trimers to treat Alport syndrome.

In vivo measurement of swallowing by monitoring thyroid cartilage movement in healthy subjects using thickened liquid samples and its comparison with sensory evaluation.

This research aimed to investigate the relationship between in vivo measurement of swallowing and sensory evaluation using thickened liquids as model foods. Healthy subjects (8 male and average 29.6-year old) participated in both tests, in which the subjects were asked to swallow the whole amount of sample (10 ml) at one time. In vivo measurement monitored thyroid cartilage movement during swallowing using a bendable pressure sensor synchronously with suprahyoid electromyography, whereas sensory evaluation measured perceived cohesiveness and adhesiveness on a visual analogue scale. Two variables from the pressure sensor analysis; activity of the thyroid cartilage and the maximum displacement of the thyroid cartilage were correlated negatively (p < .01) to perceived cohesiveness with high correlation coefficient (|r|> .9). Advantages of in vivo measurement over conventional shear rheology in assessing texture attributes perceived during swallowing were identified. PRACTICAL APPLICATIONS: This research provides food manufactures with knowledge on a novel objective method for texture measurement of fluid foods and beverages based on human physiology during swallowing. Variable from this method can work as a measure for texture design of food products to meet consumers' preference, particularly foods for dysphagia patients whose demand is increasing in this aged society although subjects of study should be expanded to these people in the future.

Curcumin Inhibits NTHi-Induced MUC5AC Mucin Overproduction in Otitis Media via Upregulation of MAPK Phosphatase MKP-1.

Otitis media (OM), characterized by the presence of mucus overproduction and excess inflammation in the middle ear, is the most common childhood infection. Nontypeable Haemophilus influenzae (NTHi) pathogen is responsible for approximately one-third of episodes of bacteria-caused OM. Current treatments for bacterial OM rely on the systemic use of antibiotics, which often leads to the emergence of multidrug resistant bacterial strains. Therefore there is an urgent need for developing alternative therapies strategies for controlling mucus overproduction in OM. MUC5AC mucin has been shown to play a critical role in the pathogenesis of OM. Here we show that curcumin derived from Curcuma longa plant is a potent inhibitor of NTHi-induced MUC5AC mucin expression in middle ear epithelial cells. Curcumin inhibited MUC5AC expression by suppressing activation of p38 MAPK by upregulating MAPK phosphatase MKP-1. Thus, our study identified curcumin as a potential therapeutic for inhibiting mucin overproduction in OM by upregulating MKP-1, a known negative regulator of inflammation.

Resveratrol suppresses NTHi-induced inflammation via up-regulation of the negative regulator MyD88 short.

Upper respiratory tract inflammatory diseases such as asthma and chronic obstructive pulmonary diseases (COPD) affect more than one-half billion people globally and are characterized by chronic inflammation that is often exacerbated by respiratory pathogens such as nontypeable Haemophilus influenzae (NTHi). The increasing numbers of antibiotic-resistant bacterial strains and the limited success of currently available pharmaceuticals used to manage the symptoms of these diseases present an urgent need for the development of novel anti-inflammatory therapeutic agents. Resveratrol has long been thought as an interesting therapeutic agent for various diseases including inflammatory diseases. However, the molecular mechanisms underlying its anti-inflammatory properties remain largely unknown. Here we show for the first time that resveratrol decreases expression of pro-inflammatory mediators in airway epithelial cells and in the lung of mice by enhancing NTHi-induced MyD88 short, a negative regulator of inflammation, via inhibition of ERK1/2 activation. Furthermore, resveratrol inhibits NTHi-induced ERK1/2 phosphorylation by increasing MKP-1 expression via a cAMP-PKA-dependent signaling pathway. Finally, we show that resveratrol has anti-inflammatory effects post NTHi infection, thereby demonstrating its therapeutic potential. Together these data reveal a novel mechanism by which resveratrol alleviates NTHi-induced inflammation in airway disease by up-regulating the negative regulator of inflammation MyD88s.

Mild electrical stimulation increases stress resistance and suppresses fat accumulation via activation of LKB1-AMPK signaling pathway in C. elegans.

Electrical current at physiological strength has been applied as a therapeutic approach for various diseases. Several of our works showed that mild electrical stimulation (MES) at 0.1-ms pulse width has positive impact on organisms. But despite the growing evidence of the beneficial effects of MES, its effects on individual animals and the molecular underpinnings are poorly understood and rarely studied. Here, we examined the effects of MES on individual animal and its mechanisms by mainly using Caenorhabditis elegans, a powerful genetic model organism. Interestingly, MES increased stress resistance and suppressed excess fat accumulation in wild-type N2 worms but not in AMPK/AAK-2 and LKB1/PAR-4 mutant worms. MES promoted the nuclear localization of transcription factors DAF-16 and SKN-1 and consequently increased the expression of anti-stress genes, whereas MES inhibited the nuclear localization of SBP-1 and suppressed the expression of lipogenic genes. Moreover, we found that MES induced the activation of LKB1/PAR4-AMPK/AAK2 pathway in C. elegans and in several mammalian cell lines. The mitochondrial membrane potential and cellular ATP level were slightly and transiently decreased by MES leading to the activation of LKB1-AMPK signaling pathway. Together, we firstly and genetically demonstrated that MES exerts beneficial effects such as stress resistance and suppression of excess fat accumulation, via activation of LKB1-AMPK signaling pathway.

Mild electrical stimulation at 0.1-ms pulse width induces p53 protein phosphorylation and G2 arrest in human epithelial cells.

Exogenous low-intensity electrical stimulation has been used for treatment of various intractable diseases despite the dearth of information on the molecular underpinnings of its effects. Our work and that of others have demonstrated that applied electrical stimulation at physiological strength or mild electrical stimulation (MES) activates the PI3K-Akt pathway, but whether MES activates other molecules remains unknown. Considering that MES is a form of physiological stress, we hypothesized that it can activate the tumor suppressor p53, which is a key modulator of the cell cycle and apoptosis in response to cell stresses. The potential response of p53 to an applied electrical current of low intensity has not been investigated. Here, we show that p53 was transiently phosphorylated at Ser-15 in epithelial cells treated with an imperceptible voltage (1 V/cm) and a 0.1-ms pulse width. MES-induced p53 phosphorylation was inhibited by pretreatment with a p38 MAPK inhibitor and transfection of dominant-negative mutants of p38, MKK3b, and MKK6b, implying the involvement of the p38 MAPK signaling pathway. Furthermore, MES treatment enhanced p53 transcriptional function and increased the expression of p53 target genes p21, BAX, PUMA, NOXA, and IRF9. Importantly, MES treatment triggered G2 cell cycle arrest, but not cell apoptosis. MES treatment had no effect on the cell cycle in HCT116 p53(-/-) cells, suggesting a dependence on p53. These findings identify some molecular targets of electrical stimulation and incorporate the p38-p53 signaling pathway among the transduction pathways that MES affects.

Insulin receptor activation through its accumulation in lipid rafts by mild electrical stress.

Insulin resistance is due to the reduced cellular response to insulin in peripheral tissues. The interaction of insulin with its receptor is the first step in insulin action and thus the identified target of insulin resistance. It has been well established that defects or mutations in the insulin receptor (IR) cause insulin resistance. Therefore, an IR activator might be a novel therapeutic approach for insulin resistance. Our previous report showed that mild electrical stress (MES) enhanced the insulin-induced signaling pathway. However, the molecular mechanism of the effect of MES remains unclear. We assessed the effect of MES, which is characterized by low-intensity direct current, on insulin signaling in vitro and in vivo. Here, we showed that MES activated the insulin signaling in an insulin-independent manner and improved insulin resistance in peripheral tissues of high fat-fed mice. Moreover, we found that MES increased the localization of IR in lipid rafts and enhanced the level of phosphorylated Akt in insulin-resistant hepatic cells. Ablation of lipid rafts disrupted the effect of MES on Akt activation. Our findings indicate that MES has potential as an activator of IR in an insulin-independent manner, and might be beneficial for insulin resistance in type 2 diabetes.

Diethyldithiocarbamate induces apoptosis in HHV-8-infected primary effusion lymphoma cells via inhibition of the NF-κB pathway.

Primary effusion lymphoma (PEL) is a subtype of B-cell lymphoma caused by human herpes virus 8/Kaposi sarcoma-associated herpes virus (HHV-8/KSHV), which is mostly found in patients with AIDS and has poor prognosis. Nuclear factor (NF)-κB pathway is constitutively activated in HHV-8-infected PEL cells and plays a crucial role in tumorigenesis. Recently, it has been shown that diethyldithiocarbamate (DDTC), an active metabolite of disulfiram, has apoptotic activity in cancer cells. Here, we investigated the effect of DDTC on PEL using a PEL mouse model generated by intraperitoneal injection of BC-3 cells, a PEL cell line. DDTC ameliorated the symptoms of PEL in these mice, such as development of ascites, splenomegaly and increase of body weight, in comparison with PBS-treated controls. Moreover, we determined in vitro that DDTC suppressed the constitutively activated NF-κB pathway in BC-3 cells. Methylthiotetrazole assay revealed that the cell proliferation of various PEL cell lines was significantly suppressed by the treatment of DDTC. DDTC also induced the expression of cleaved caspase-3, an apoptosis marker, whereas the addition of Q-VD-OPh, a pan-caspase inhibitor, inhibited cell apoptosis induced by DDTC treatment. Together, our results indicated that DDTC induces apoptosis via inhibition of the NF-κB signaling pathway in HHV-8-infected PEL cells. This study suggests the potential use of DDTC as a therapeutic approach for PEL.

Glucose uptake in rat skeletal muscle L6 cells is increased by low-intensity electrical current through the activation of the phosphatidylinositol-3-OH kinase (PI-3K) / Akt pathway.

Activation of Akt by insulin is transmitted via phosphatidylinositol-3-OH kinase (PI-3K) and enhances glucose uptake. The PI-3K/Akt signaling is diminished in insulin resistance. Thus, approaches that activate PI-3K/Akt signaling leading to improved glucose uptake may ameliorate hyperglycemia. Here we showed that low-intensity electrical current or mild electrical stimulation (MES) activated the PI-3K/Akt signaling and increased the glucose uptake in rat skeletal muscle (L6) cells. The glucose uptake enhanced by MES in muscle cells, the major cells involved in glucose disposal, suggests MES may have a possible beneficial effect on hyperglycemia.