DAMP Molecule S100A9 Acts as a Molecular Pattern to Enhance Inflammation during Influenza A Virus Infection: Role of DDX21-TRIF-TLR4-MyD88 Pathway

Pathogen-associated molecular patterns (PAMPs) trigger host immune response by activating pattern recognition receptors like toll-like receptors (TLRs). However, the mechanism whereby several pathogens, including viruses, activate TLRs via a non-PAMP mechanism is unclear. Endogenous “inflammatory mediators” called damage-associated molecular patterns (DAMPs) have been implicated in regulating immune response and inflammation. However, the role of DAMPs in inflammation/immunity during virus infection has not been studied. We have identified a DAMP molecule, S100A9 (also known as Calgranulin B or MRP-14), as an endogenous non-PAMP activator of TLR signaling during influenza A virus (IAV) infection. S100A9 was released from undamaged IAV-infected cells and extracellular S100A9 acted as a critical host-derived molecular pattern to regulate inflammatory response outcome and disease during infection by exaggerating pro-inflammatory response, cell-death and virus pathogenesis. Genetic studies showed that the DDX21-TRIF signaling pathway is required for S100A9 gene expression/production during infection. Furthermore, the inflammatory activity of extracellular S100A9 was mediated by activation of the TLR4-MyD88 pathway. Our studies have thus, underscored the role of a DAMP molecule (i.e. extracellular S100A9) in regulating virus-associated inflammation and uncovered a previously unknown function of the DDX21-TRIF-S100A9-TLR4-MyD88 signaling network in regulating inflammation during infection.     

Circulating 4-hydroxynonenal-protein thioether adducts assessed by gas chromatography-mass spectrometry are increased with disease progression and aging in spontaneously hypertensive rats

Oxidative stress has been implicated in numerous degenerative diseases of aging, including heart diseases. However, there is still a need to identify biomarkers of oxidative stress-related events, such as protein modification by the lipid peroxidation product 4-hydroxynonenal (HNE) in these diseases in humans. The objective of this study was to assess if circulating levels of HNE-protein adducts (i) can be assessed with precision by GCMS and (ii) vary with disease progression and aging in a model of cardiomyopathy that displays enhanced oxidative stress, namely the spontaneously hypertensive rats (SHR). We modified a previously published isotope dilution GCMS method that quantifies HNE and its inactive metabolite, 1,4-dihydroxynonene (DHN), bound to thiol proteins following treatment with NaB(2)H(4) and Raney nickel, to increase its sensitivity (20-fold), precision, and robustness. Levels of these adducts were measured in blood and plasma collected from SHR and control Wistar rats at 7, 15, 22, and 30 weeks of age. Levels of protein-bound HNE, which were quantitated with good precision in the nanomolar range in blood, but not in plasma, were significantly increased by disease (SHR) and age (P < 0.0001 for both). Compared to Wistar rats, SHR showed greater blood levels of HNE-protein adducts at 22 and 30 weeks. Levels of protein-bound DHN, which were detected in blood and in plasma, were not affected by disease or age. Collectively, the results of this study conducted in an animal model of cardiomyopathy demonstrate that changes in blood HNE-protein thioether adducts with disease progression and aging can be assessed with good precision by the described GCMS method. This method may prove to be useful in evaluating the occurrence and impact of oxidative stress-related events involving bioactive HNE in heart diseases and aging in humans.     

Two generation reproduction study of ethylbenzene by inhalation in Crl-CD rats

BACKGROUND: This study was conducted to evaluate the potential adverse effects of ethylbenzene (EB) on reproductive capability from whole-body inhalation exposure of F0 and F1 parental animals. METHODS: Four groups of Crl:CD(SD)IGS BR rats (30/sex/group for F0 and 25/sex/group for F1) were exposed to 0, 25, 100, and 500 ppm EB for 6 hr/day for at least 70 consecutive days before mating. Inhalation exposure for the F0 and F1 females continued throughout mating, gestation through gestation day (GD) 20, and lactation days (LD) 5-21. On LD 1-4, females received EB in corn oil via oral gavage at dose levels of 26, 90, and 342 mg/kg/day (divided into three equal doses, approximately 2 hr apart), as calculated from a physiologically-based pharmacokinetic (PBPK) model to provide similar maternal blood area-under-concentration (AUC) as provided by inhalation. Pups were weaned on postnatal day (PND) 21 and exposure of the F1 generation started on PND 22. Estimates of internal exposure were determined by measuring EB concentrations in blood collected from F1 dams (4/group) and their culled pups 1 hr after the last gavage dose on PND 4. On PND 22, blood was collected from these same F1 dams and their weanlings for EB analysis 1 hr after a 6-hr inhalation exposure. The remainder of the F2 generation was not directly exposed. RESULTS: EB exposure did not affect survival or clinical observations. Male rats in the 500 ppm group in both generations gained weight more slowly than the controls. There were no indications of adverse effects on reproductive performance in either generation. Male and female mating and fertility indices, pre-coital intervals, spermatogenic endpoints, ovarian follicle counts, reproductive organ weights, lengths of estrous cycle and gestation, live litter size, pup weights, developmental landmarks, and postnatal survival were unaffected. No adverse exposure-related macroscopic pathology was noted at any level. CONCLUSIONS: Increased liver weights were found in the animals exposed to 500 ppm. F1 maternal whole blood EB concentrations of 0.49, 3.51, or 18.28 mg/L were found 1 hr after administration of a composite oral dose of 26, 90, or 342 mg/kg/day, respectively, but no detectable EB was found in blood samples of their F2 PND 4 culled pups. F1 maternal mean whole blood EB levels 1 hr after a 6-hr inhalation exposure on postpartum day (PPD) 22 was 0.11 mg/L (25 ppm), 0.56 mg/L (100 ppm), and 11 mg/L (500 ppm). For the offspring exposed with their dams on PND 22, F2 pup blood EB concentrations ranged from 0.017-0.039 mg/L (25 ppm), 0.165-0.465 mg/L (100 ppm), and 8.82-15.74 mg/L (500 ppm). Because decreased weight gain in the 500 ppm males was transient and no histopathological changes were associated with the increased liver weights in the 500 ppm male and female groups, these changes were not considered adverse. Therefore, for parental systemic toxicity, 100 ppm was considered a NOEL and 500 ppm a NOAEL in this study. The 500 ppm exposure concentration was considered a NOAEL for F0 and F1 reproductive toxicity and offspring developmental endpoints.     

Random Mutagenesis of Clostridium cellulolyticum by Using a Tn1545 Derivative

Further understanding of the plant cell wall degradation system of Clostridium cellulolyticum and the possibility of metabolic engineering in this species highlight the need for a means of random mutagenesis. Here, we report the construction of a Tn1545-derived delivery tool which allows monocopy random insertion within the genome.The economic feasibility and sustainability of lignocellulosic ethanol production are dependent on the development of robust microorganisms which can efficiently degrade and/or convert plant biomass to ethanol (5). The anaerobic, mesophilic, Gram-positive bacterium Clostridium cellulolyticum is a candidate microorganism, as it is capable of hydrolyzing plant cell wall polysaccharides and fermenting the hydrolysis products to ethanol and other metabolites (7). C. cellulolyticum achieves this efficient hydrolysis by using multiprotein extracellular enzymatic complexes, termed cellulosomes (13). As plant cell walls consist of several intertwined heterogeneous polymers, primarily composed of cellulose, hemicellulose, and pectin, cellulosomes contain many subunits (cellulosomal enzymes) with diverse and complementary enzymatic properties (2). Thus, this model organism is also a good candidate for the development of novel and efficient cellulases and hemicellulases for the saccharification of plant biomass.Gene transfer has been successfully carried out in C. cellulolyticum (8, 12). This possibility has allowed the in vivo function of cellulosomal enzymes in C. cellulolyticum to be examined by overexpression (9) or down expression (11) of targeted genes. However, random mutagenesis of the entire chromosome and screening of mutants to identify key components for plant cell wall degradation have never been described. Conjugative transfer of Tn1545 from Enterococcus faecalis to C. cellulolyticum has been described but is limited by low transfer frequency and poor reproducibility (8). To improve transposon mutagenesis of C. cellulolyticum, we exploited the two-plasmid Tn1545 delivery system described by Trieu-Cuot et al. (15). In this system, the Tn916 integrase-encoding gene is carried by an expression vector, whereas the attachment site of Tn1545 is carried by a suicide vector. Tn916 and Tn1545 being closely related (4), integration of the Tn1545 derivative occurs in the genome after transformation of the strain with both vectors (15).     

Taxonomic status of Cuscuta nevadensis and C. veatchii (Convolvulaceae) in North America

The taxonomy ofCuscuta nevadensis andC. veatchii is investigated.Cuscuta nevadensis is more closely related toC. veatchii andC. denticulata than toC. salina, and the former two taxa are accepted as species. A summary of relevant taxonomic and biological information is provided, including synonymy, distribution and ecology, keys, and a comparison of the morphology of flowers and seeds are examined. The morphological basis of vivipary inC. nevadensis is discussed. The status ofCuscuta vivipara, an invalid name in recent use, is clarified.     

Electrotransformation studies in Clostridium cellulolyticum

Electropermeabilization of Clostridium cellulolyticum was optimized using ATP leakage assays. Electrotransformation was then performed under optimized conditions (6 to 7.5 kV cm⁻¹ field strength applied during 5 ms to a mixture containing methylated plasmids and late exponential phase cell suspensions (10 molecules:1 cell) in a sucrose-containing buffer). Transformants were only obtained when 7 or 7.5 kV cm⁻¹ pulses were applied. Transformation efficiencies evaluated from the growth curves of transformed cells were between 10⁵ and 10⁷ transformants per microgram of plasmid DNA for five different replicon-based plasmids. Restriction nuclease digestion patterns of pJIR418 purified from transformed Clostridia and Escherichia coli were indistinguishable, indicating that heterologous DNA was structurally stable in the Clostridium strain. Copy numbers of 130, 70 and 10 were estimated from purification yield for pCTC1, pKNT19 and pJIR418, respectively. Journal of Industrial Microbiology & Biotechnology (2001) 27, 271–274. Received 12 September 2000/ Accepted in revised form 25 November 2000     

Comparison of infant care in family groups of the common marmoset (Callithrix jacchus) and the cotton-top tamarin (Saguinus oedipus)

The involvement of parents and siblings in infant care in similarly composed groups of common marmosets (Callithrix jacchus) and cotton-top tamarins (Saguinus oedipus) was compared during the infants' first 8 weeks of life. The results indicate an earlier infant independence in C. jacchus than in S. oedipus due primarily to a more frequent rejection of carried infants in C. jacchus. There was no species difference in extent of maternal involvment in carrying infants. However, S. oedipus fathers carried infants significantly more often during weeks 5–8 than did C. jacchus fathers. Siblings were generally more involved in infant care at an earlier infant age in C. jacchus than in S. oedipus.     

Influence of a cysteine prodrug, L-2-oxothiazolidine-4-carboxylic acid, on the urinary elimination of mercapturic acids of ethylene oxide, dibromoethane, and acrylonitrile: a dose-effect study

Metabolic disposition of ethylene oxide, dibromoethane, and acrylonitrile in rats after acute exposure was studied by examining the relationship between dose and urinary metabolites, and by establishing the influence of a glutathione precursor, L-2-oxothiazolidine-4-carboxylic acid (OTCA), on the above relationship. Respective urinary metabolites, hydroxyethylmercapturic acid, cyanoethylmercapturic acid, thiocyanate, and ethylene glycol, were quantified to estimate the extent to which each compound was metabolized. The animals were given either ethylene oxide (0.34, 0.68, or 1.36 mmol/kg), dibromoethane (0.2, 0.4, or 0.6 mmol/kg), or acrylonitrile (0.10, 0.38, or 0.76 mmol/kg). Urine samples were collected at 24 h. The metabolic biotransformation of all three chemicals to their respective mercapturic acids was strongly indicative of saturable metabolism. Administration of OCTA (4-5 mmol/kg) enhanced gluthathione availability and increased excretion of urinary mercapturic acids at the higher doses of the chemicals. This study indicates that OTCA increases the capacity for detoxification via the glutathione pathway thereby partially correcting the nonlinearity between the administered dose of ethylene oxide, dibromoethane, and acrylonitrile and the amount of certain urinary metabolites.     

Social influences on sexual maturation of female Saguinus oedipus oedipus

The hypothesis tested was that Saguinus oedipus oedipus females housed with adult males would mature, sexually, at an earlier age than females remaining in their natal family groups. Six females were housed with strange, unrelated males. Five females remained in their natal groups. Blood samples were taken twice weekly, and the plasma was assayed for progesterone. Sexual maturation was operationally defined as that age at which plasma progesterone levels became consistently detectable. Females housing with males did mature at an earlier age than females remaining in their natal groups. In addition, it was noted that the presence or absence of a healthy, reproductive mother in the natal group was not related to the daughter's maturation age. However, whether the natal group, as a whole, inhibited maturation of young females, or an unrelated male accelerated maturation, or both, remains unknown.