Vaccination with Klebsiella pneumoniae-derived extracellular vesicles protects against bacteria-induced lethality via both humoral and cellular immunity

The emergence of multidrug-resistant Klebsiella pneumoniae highlights the need to develop preventive measures to ameliorate Klebsiella infections. Bacteria-derived extracellular vesicles (EVs) are spherical nanometer-sized proteolipids enriched with outer membrane proteins. Gram-negative bacteria-derived EVs have gained interest for use as nonliving complex vaccines. In the present study, we evaluated whether K. pneumoniae-derived EVs confer protection against bacteria-induced lethality. K. pneumoniae-derived EVs isolated from in vitro bacterial culture supernatants induced innate immunity, including the upregulation of co-stimulatory molecule expression and proinflammatory mediator production. EV vaccination via the intraperitoneal route elicited EV-reactive antibodies and interferon-gamma-producing T-cell responses. Three vaccinations with the EVs prevented bacteria-induced lethality. As verified by sera and splenocytes adoptive transfer, the protective effect of EV vaccination was dependent on both humoral and cellular immunity. Taken together, these findings suggest that K. pneumoniae-derived EVs are a novel vaccine candidate against K. pneumoniae infections.     

Structure-toxicity relationships for aliphatic compounds encompassing a variety of mechanisms of toxic action to Vibrio fischeri

QSARs based upon the logarithm of the octanol-water partition coefficient, log P, and energy of the lowest unoccupied molecular orbital, ELUMO were developed to model the toxicity of aliphatic compounds to the marine bacterium Vibrio fischeri. Statistically robust, hydrophobic-dependent QSARs were found for chloroalcohols and haloacetonitriles. Modelling of the toxicity of the haloesters and the diones required the use of terms to describe both hydrophobicity and electrophilicity. The differences in intercepts, slopes, and fit of these models suggest different electrophilic mechanisms occur between classes, as well as within the diones and haloesters. In order to model globally the toxicity of aliphatic compounds to V. fischeri, all the data determined in this study were combined with those determined previously for alkanones, alkanals, and alkenals. A highly predictive two-parameter QSAR [pT15 = 0.760(log P) - 0.625(ELUMO) - 0.466; n = 63, s = 0.462, r2 = 0.846, F = 171, Pr > F = 0.0001] was developed for the combined data that models across classes and is independent of mechanisms of action. The toxicity of these compounds to V. fischeri compares well to the toxicity (50% population growth inhibition) to the ciliate Tetrahymena pyriformis (r2 = 0.850).     

Ultraviolet action spectra for DNA dimer induction, lethality, and mutagenesis in Escherichia coli with emphasis on the UVB region

Abstract —Ultraviolet (UV) action spectra were obtained for lethality and mutagenesis (reversion to tryptophan independence) in Escherichia coli WP2s for wavelengths 254–405 nm with detailed analysis in the UVB region (290–320 nm). Parallel chemical assay yields of pyrimidine dimers in DNA of E. coli RT4 were determined at the same wavelengths. Spectral regions isolated from a Xe arc and resonance lines from a high-pressure Hg-Xe arc lamp were both used for irradiation. In all cases, precise energy distributions throughout the isolated Xe bands regions were defined.
Lethality, mutagenesis, and dimer induction all decreased in efficiency in a similar fashion as the wavelengths of the radiation increased. Between 300 and 320 nm, all characteristics measured showed differences of about two and a half orders of magnitude. Between these wavelengths, the values of the three end points used either coincide with or parallel the absorption spectrum of DNA. The mutagenesis action spectrum coincides closely with the absorption spectrum of DNA. The lethality spectrum is closely parallel to the mutagenicity spectrum; the points, however, consistently occur at about 2 nm longer wavelengths. A calculation derived from the slope of the UVB spectra reveals that a 1-nm shift of the solar UV spectrum to shorter wavelengths would result in a 35% increase in its mutagenic potential. At 325 nm, both biological action spectra show sharp decreases in slope. In addition, above 325 nm the spectra for lethality. mutagenicity, and dimer formation diverge sharply; lethalities at these UVA wavelengths were approximately tenfold greater relative to mutagenicity than at shorter wavelengths. The relative yield of dimer formation by 365 nm radiation is intermediate between the yields for lethality and mutagenesis.     

Transcellular transport of low density lipoprotein through cultured newborn rat skin epidermal cell monolayer

Epidermal cells from newborn rat skin were cultured on type IV collagen-coated Millipore filter, and the transport of low density lipoprotein (LDL) labeled with Rhodamine B isothiocyanate (RB-LDL) through the cultured cell layer was examined. The transport of RB-LDL was dependent on temperature and biological energy. The transport was low at 17 degrees C, but above 20 degrees C, it became high with increase in temperature up to 37 degrees C. The transport was markedly inhibited by the energy inhibitors 2-deoxyglucose and NaN3. Furthermore, the transport was saturable at the RB-LDL concentration of about 300 micrograms/ml and the activation energy of the transport was determined as 104.6 kJ/mol. No degradation product of LDL (apoprotein B) was observed during LDL transport through the cultured cell layer. The transport of RB-LDL through skin epidermal cells in culture is suggested to be mediated by transcytotic vesicles, but not by endocytosis and exocytosis via the lysosomal system, nor through cellular junctions.     

Coptidis rhizoma extract protects against cytokine-induced death of pancreatic beta-cells through suppression of NF-kappaB activation

We demonstrated previously that Coptidis rhizoma extract (CRE) prevented S-nitroso-N-acetylpenicillamine-induced apoptotic cell death via the inhibition of mitochondrial membrane potential disruption and cytochrome c release in RINm5F (RIN) rat insulinoma cells. In this study, the preventive effects of CRE against cytokine-induced beta-cell death was assessed. Cytokines generated by immune cells infiltrating pancreatic islets are crucial mediators of beta-cell destruction in insulin-dependent diabetes mellitus. The treatment of RIN cells with IL-1beta and IFN-gamma resulted in a reduction of cell viability. CRE completely protected IL-1beta and IFN-gamma-mediated cell death in a concentration-dependent manner. Incubation with CRE induced a significant suppression of IL-1beta and IFN-gamma-induced nitric oxide (NO) production, a finding which correlated well with reduced levels of the iNOS mRNA and protein. The molecular mechanism by which CRE inhibited iNOS gene expression appeared to involve the inhibition of NF-kappaB activation. The IL-1beta and IFN-gamma-stimulated RIN cells showed increases in NF-kappaB binding activity and p65 subunit levels in nucleus, and IkappaB alpha degradation in cytosol compared to unstimulated cells. Furthermore, the protective effects of CRE were verified via the observation of reduced NO generation and iNOS expression, and normal insulin-secretion responses to glucose in IL-1beta and IFN-gamma-treated islets.     

Flame retardant polypropylene through the joint action of sepiolite and polyamide 6

The influence of the incorporation of polyamide-6 (PA) and natural sepiolite nanoparticles on both the thermal degradation and fire behaviour of polypropylene (PP) matrix has been investigated by thermogravimetric analysis (TGA) and mass loss calorimetry. For that purpose, PP/PA blends and nanocomposites thereof were prepared by melt processing. TGA results evidenced that the use of maleic anhydride grafted-polypropylene (MA-g-PP) as compatibilizer led to a significant improvement in thermal stability under air. Such improvement was linked to the formation of a char layer preventing the thermo-oxidative degradation of PP. Interestingly, the thermal resistance of this char layer was further improved by adding 5 wt% of natural sepiolite leading to important increase of time to ignition and reduction of peak of heat release rate (pHRR) during mass loss calorimeter test.     

Endothelin 1 protects HN33 cells from serum deprivation-induced neuronal apoptosis through Ca(2+)-PKCalpha-ERK pathway

Endothelins (ETs), which were originally found to be potent vasoactive transmitters, were known to be implicated in nervous system, but the mode of mechanism remains unclear. ETs (ET-1, ET-2, and ET-3) were added to HN33 (mouse hippocampal neuron chi neuroblastoma) cells. Among the three types of ET, only ET-1 increased the intracellular calcium levels in a PLC dependent manner with the induction of ERK 1/2 activation. As the result of ET-1 exposure, the survival rate of HN33 cells and the PKCalpha translocation into the plasma membrane were increased. We suggest that ET-1 participated in the neuroprotective effect involving the calcium-PKCalpha-ERK1/2 pathway.     

Capillary zone electrophoresis for the quantitation of oligosaccharides formed through the action of chitinase.

Capillary zone electrophoresis with fluorescence detection was used to analyze the products formed by chitinase acting on N-acetylchitooligosaccharide-fluorescent conjugates. Six oligosaccharides of the structure [N-acetylglucosamine(1----4)]n (where n = 1-6) were conjugated to 7-amino-1,3-naphthalene disulfonic acid by reductive amination. Each oligosaccharide-fluorescent conjugate was purified by preparative gradient polyacrylamide gel electrophoresis, semi-dry electrotransfer to a positively-charged nylon membrane and recovered by washing the membrane with salt solution. The products formed by treating each oligosaccharide-fluorescent conjugate with chitinase were analyzed by capillary zone electrophoresis. The chitinase treatment hexasaccharide-fluorescent conjugate was also examined kinetically to study the action pattern of this enzyme.     

Paecilomyces variotii extract increases lifespan and protects against oxidative stress in Caenorhabditis elegans through SKN-1, but not DAF-16

A natural extract from Paecilomyces variotii (P. variotii extract, PVE), an endophytic fungus, has been used widely to improve agricultural crop performance and control multiple plant pathogens. Most recent studies focused on its application as a plant growth promoter, while relatively few studies have been reported on the antioxidant potential in vivo and the underlying mechanism. The present study was designed to determine the antioxidant activities of PVE and its mechanisms using Caenorhabditis elegans. Results showed that, compared to the solvent control, PVE at 1.0, 10 and 100 ng/mL significantly extended the lifespan of C. elegans by 36.60%, 59.80% and 53.30%, respectively. PVE at 10 ng/mL consistently promoted nematodes growth, but all treatments did not influence nematode fecundity, locomotion behavior, and pharyngeal pumping. Furthermore, PVE at the three tested concentrations significantly reduced accumulation of reactive oxygen species (ROS), lipofuscin, lipid and malondialdehyde (MDA) content, meanwhile significantly promoted activities of superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST) in the nematodes. Compared with the solvent control, PVE up-regulated gene expression of skn-1, mev-1, sod-3, and daf-2, but significantly down-regulated the expression of nhr-49 and daf-16. Further evidence revealed that PVE at the three concentrations significantly promoted nuclear localization of SKN-1, but not affected that of DAF-16, indicating the complex roles of DAF-16 and SKN-1 in stress resistance and longevity regulation. Overall, our results demonstrated that SKN-1 played a critical role in increasing lifespan of C. elegans and protecting the nematodes from oxidative stress, independent of DAF-16.     

Mechanisms of antioxidant action: Formation of antioxidant adducts with rubbers through sulphur by a mechanochemical procedure

It is shown that antioxidants containing a thiol group can be mechanochemically reacted with both natural rubber (NR) and styrene-butadiene-rubber (SBR). Oxygen inhibits the reaction and the optimum temperature is in the region of 70°C. Increase in the antioxidant concentration in the rubber leads to increasing levels of adduct formation. Evidence is presented to show that although a substantial part of the adduct is formed during mechanochemical treatment, about 20% of the total binding occurs during the vulcanisation reaction.     

MRI-guided neutron capture therapy by use of a dual gadolinium/boron agent targeted at tumour cells through upregulated low-density lipoprotein transporters

The upregulation of low-density lipoprotein (LDL) transporters in tumour cells has been exploited to deliver a sufficient amount of gadolinium/boron/ligand (Gd/B/L) probes for neutron capture therapy, a binary chemio-radiotherapy for cancer treatment. The Gd/B/L probe consists of a carborane unit (ten B atoms) bearing an aliphatic chain on one side (to bind LDL particles), and a Gd(III)/1,4,7,10-tetraazacyclododecane monoamide complex on the other (for detection by magnetic resonance imaging (MRI)). Up to 190 Gd/B/L probes were loaded per LDL particle. The uptake from tumour cells was initially assessed on cell cultures of human hepatoma (HepG2), murine melanoma (B16), and human glioblastoma (U87). The MRI assessment of the amount of Gd/B/L taken up by tumour cells was validated by inductively coupled plasma-mass-spectrometric measurements of the Gd and B content. Measurements were undertaken in vivo on mice bearing tumours in which B16 tumour cells were inoculated at the base of the neck. From the acquisition of magnetic resonance images, it was established that after 4-6 hours from the administration of the Gd/B/L-LDL particles (0.1 and 1 mmol kg(-1) of Gd and (10)B, respectively) the amount of boron taken up in the tumour region is above the threshold required for successful NCT treatment. After neutron irradiation, tumour growth was followed for 20 days by MRI. The group of treated mice showed markedly lower tumour growth with respect to the control group.     

Levan polysaccharide from Erwinia herbicola protects osteoblast cells against lipopolysaccharide-triggered inflammation and oxidative stress through regulation of ChemR23 for prevention of osteoporosis

Osteoblast cell injury is a type of degenerative disorder characterized by osteolysis. Levan polysaccharide is an active component of Erwinia herbicola, which shows potential anti-inflammatory and antioxidant properties. However, the protective effects of levan on lipopolysaccharide (LPS)-induced inflammatory and oxidative stress injury in osteoblast cells as an in vitro model of osteolysis remain largely unknown. The present study aimed to explore the functions of levan in LPS-triggered inflammation and oxidative stress in osteoblast cells (MC3T3-E1). The protective effects of levan on LPS-induced inflammatory and oxidative stress responses in MC3T3-E1 cells were assessed by quantification of superoxide dismutase (SOD) and catalase (CAT) activity as well as enzyme-linked immunosorbent assay (ELISA) for determination of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). Also, the key signaling pathway of ChemR23 was determined by qPCR analysis. Results showed that levan significantly alleviated LPS-induced deactivation of SOD and CAT activity. Levan also downregulated the expression of IL-6, TNF-α, and ChemR23 at mRNA level. These findings indicated that levan may protect MC3T3-E1 cells against LPS-triggered oxidative stress, and inflammation via regulation of ChemR23. This data may provide a potential basis for further clinical investigation of levan in the prevention and treatment of LPS-triggered bone resorption and osteolysis.     

CD36 signaling inhibits the translation of heat shock protein 70 induced by oxidized low density lipoprotein through activation of peroxisome proliferators-activated receptor ��

Oxidized LDL (OxLDL), a causal factor in atherosclerosis, induces the expression of heat shock proteins (Hsp) in a variety of cells. In this study, we investigated the role of CD36, an OxLDL receptor, and peroxisome proliferator-activated receptor γ (PPARγ) in OxLDL-induced Hsp70 expression. Overexpression of dominant-negative forms of CD36 or knockdown of CD36 by siRNA transfection increased OxLDL-induced Hsp70 protein expression in human monocytic U937 cells, suggesting that CD36 signaling inhibits Hsp70 expression. Similar results were obtained by the inhibition of PPARγ activity or knockdown of PPARγ expression. In contrast, overexpression of CD36, which is induced by treatment of MCF-7 cells with troglitazone, decreased Hsp70 protein expression induced by OxLDL. Interestingly, activation of PPARγ through a synthetic ligand, ciglitazone or troglitazone, decreased the expression levels of Hsp70 protein in OxLDL-treated U937 cells. However, major changes in Hsp70 mRNA levels were not observed. Cycloheximide studies demonstrate that troglitazone attenuates Hsp70 translation but not Hsp70 protein stability. PPARγ siRNA transfection reversed the inhibitory effects of troglitazone on Hsp70 translation. These results suggest that CD36 signaling may inhibit stress-induced gene expression by suppressing translation via activation of PPARγ in monocytes. These findings reveal a new molecular basis for the anti-inflammatory effects of PPARγ.     

Transport of alkali-metal picrates through liquid membranes: Coupled action of w/o microemulsion droplets and lipophilic crown-ether carriers

The transport of potassium and sodium picrates is measured by a liquid membrane technique in the presence of two mobile carriers: water-in-oil microemulsion droplets and lipophilic crown-ethers. The fluxes are compared for two cases: (i) the crown-ether (dicyclohexano-18-crown-6 is assumed to be independent of the microemulsion droplet and (ii the crown-ether (dodecano- 18-crown-6) is assumed to be anchored in the droplet. A lower flux is observed when the microemulsion droplet and the crown-ether diffuse as a single entity. These results suggest that larger synergistic effects are obtained when the extractant can diffuse independently of the microemulsion droplet.     

Ethanol and halothane differently modulate HLA class I and class II oligomerization. A new look at the mode of action of anesthetic agents through fluorescence spectroscopy

The field of research considering the working mechanism of anesthetic agents is a complex one and the site or sites of action of general anesthetics are yet to be elucidated. Through the years, on the molecular level, the discussion has shifted from the lipid theories to the more specific interaction with the proteins responsible for the signal transduction. While this approach led to several models, they offer, at best, partial explanations for the observed phenomena. Anesthetic agents interact with many systems, of which the neuronal is best studied, leaving interaction with the immune defense system relatively unexplored. In this study we focus on the interaction of ethanol and halothane with the co-localization on the membrane of HLA I and II molecules. We show that ethanol tends to randomize the distribution of HLA I and II molecules, while halothane increases the clustering of HLA I proteins. The notion that anesthetics modulate cell function by disrupting clustering and thereby promoting a random distribution is a novel approach that may explain the general involvement of many systems during exposition to anesthetic drugs. In this study we show the disturbance of co-localization of molecules that may form a functional network. The relevance of this finding depends on the importance of these networks for extracellular and intracellular processes.