Cellular damage, including wounding, drives C. elegans stress-induced sleep.

Across animal phyla, sleep is associated with increased cellular repair, suggesting that cellular damage may be a core component of sleep pressure. In support of this notion, sleep in the nematode Caenorhabditis elegans can be triggered by damaging conditions, including noxious heat, high salt, and ultraviolet light exposure. It is not clear, however, whether this stress-induced sleep (SIS) is a direct consequence of cellular damage, or of a resulting energy deficit, or whether it is triggered simply by the sensation of noxious conditions. Here, we show that thermosensation is dispensable for heat-induced sleep, that osmosensation is dispensable for salt-induced sleep, and that wounding is also a sleep trigger, together indicating that SIS is not triggered by sensation of noxious environments. We present evidence that genetic variation in cellular repair pathways impacts sleep amount, and that SIS involves systemic monitoring of cellular damage. We show that the low-energy sensor AMP-activated protein kinase (AMPK) is not required for SIS, suggesting that energy deficit is not the primary sleep trigger. Instead, AMPK-deficient animals display enhanced SIS responses, and pharmacological activation of AMPK reduces SIS, suggesting that ATP-dependent repair of cellular damage mitigates sleep pressure.

Structural basis for inhibition of TLR2 by staphylococcal superantigen-like protein 3 (SSL3).

Toll-like receptors (TLRs) are crucial in innate recognition of invading micro-organisms and their subsequent clearance. Bacteria are not passive bystanders and have evolved complex evasion mechanisms. Staphylococcus aureus secretes a potent TLR2 antagonist, staphylococcal superantigen-like protein 3 (SSL3), which prevents receptor stimulation by pathogen-associated lipopeptides. Here, we present crystal structures of SSL3 and its complex with TLR2. The structure reveals that formation of the specific inhibitory complex is predominantly mediated by hydrophobic contacts between SSL3 and TLR2 and does not involve interaction of TLR2-glycans with the conserved Lewis(X) binding site of SSL3. In the complex, SSL3 partially covers the entrance to the lipopeptide binding pocket in TLR2, reducing its size by ∼50%. We show that this is sufficient to inhibit binding of agonist Pam2CSK4 effectively, yet allows SSL3 to bind to an already formed TLR2-Pam2CSK4 complex. The binding site of SSL3 overlaps those of TLR2 dimerization partners TLR1 and TLR6 extensively. Combined, our data reveal a robust dual mechanism in which SSL3 interferes with TLR2 activation at two stages: by binding to TLR2, it blocks ligand binding and thus inhibits activation. Second, by interacting with an already formed TLR2-lipopeptide complex, it prevents TLR heterodimerization and downstream signaling.

The Role of Typhoid Toxin in Salmonella Typhi Virulence
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Unlike many of the nontyphoidal Salmonella serovars such as S. Typhimurium that cause restricted gastroenteritis, Salmonella Typhi is unique in that it causes life-threatening typhoid fever in humans. Despite the vast difference in disease outcomes that S. Typhi and S. Typhimurium cause in humans, there are few genomic regions that are unique to S. Typhi. Of these regions, the most notable is the small locus encoding typhoid toxin, an AB toxin that has several distinct characteristics that contribute to S. Typhi's pathogenicity. As a result, typhoid toxin and its role in S. Typhi virulence have been studied in an effort to gain insight into potential treatment and prevention strategies. Given the rise of multidrug-resistant strains, research in this area has become increasingly important. This article discusses the current understanding of typhoid toxin and potential directions for future research endeavors in order to better understand the contribution of typhoid toxin to S. Typhi virulence.

Implications of microbiota and bile acid in liver injury and regeneration.

Studies examining the mechanisms by which the liver incurs injury and then regenerates usually focus on factors and pathways directly within the liver, neglecting the signaling derived from the gut-liver axis. The intestinal content is rich in microorganisms as well as metabolites generated from both the host and colonizing bacteria. Through the gut-liver axis, this complex "soup" exerts an immense impact on liver integrity and function. This review article summarizes data published in the past 30 years demonstrating the signaling derived from the gut-liver axis in relation to liver injury and regeneration. Due to the intricate networks of implicated pathways as well as scarcity of available mechanistic data, it seems that nutrigenomic, metabolomics, and microbiota profiling approaches are warranted to provide a better understanding regarding the interplay and impact between nutrition, bacteria, and host response in influencing liver function and healing. Therefore elucidating the possible molecular mechanisms that link microbiota alteration to host physiological response and vice versa.

Septic acute kidney injury: molecular mechanisms and the importance of stratification and targeting therapy.

The most common cause of acute kidney injury (AKI) in hospitalized patients is sepsis. However, the molecular pathways and mechanisms that mediate septic AKI are not well defined. Experiments performed over the past 20 years suggest that there are profound differences in the pathogenesis between septic and ischemic AKI. Septic AKI often occurs independently of hypoperfusion, and is mediated by a concomitant pro- and anti-inflammatory state that is activated in response to various pathogen-associated molecular patterns, such as endotoxin, as well as damage-associated molecular patterns. These molecular patterns are recognized by Toll-like receptors (TLRs) found in the kidney, and effectuate downstream inflammatory pathways. Additionally, apoptosis has been proposed to play a role in the pathogenesis of septic AKI. However, targeted therapies designed to mitigate the above aspects of the inflammatory state, TLR-related pathways, and apoptosis have failed to show significant clinical benefit. This failure is likely due to the protean nature of septic AKI, whereby different patients present at different points along the immunologic spectrum. While one patient may benefit from targeted therapy at one end of the spectrum, another patient at the other end may be harmed by the same therapy. We propose that a next important step in septic AKI research will be to identify where patients lie on the immunologic spectrum in order to appropriately target therapies at the inflammatory cascade, TLRs, and possibly apoptosis.

Low-dose endotoxin induces inflammation by selectively removing nuclear receptors and activating CCAAT/enhancer-binding protein δ.

Subclinical levels of circulating endotoxin are associated with the pathogenesis of diverse human inflammatory diseases, by mildly inducing the expression of proinflammatory mediators. In this study, we examined the molecular mechanism responsible for the effect of low-dose LPS in macrophages. In contrast to high-dose LPS, which activates NF-κB and induces the robust expression of proinflammatory mediators, we observed that low-dose LPS failed to activate NF-κB. Instead, it selectively activated C/EBPδ and removed nuclear repressors, including peroxisome proliferator-activated receptor α and retinoic acid receptor α, enabling a mild and leaky expression of proinflammatory mediators. The effect of low-dose LPS required IRAK-1, which interacts with and acts upstream of IκB kinase ε to contribute to LPS-mediated induction of C/EBPδ and proinflammatory mediators. Additionally, mice fed a high-fat diet acquired elevated levels of endotoxin and proinflammatory mediators in an IRAK-1-dependent fashion. Taken together, these data reveal a distinct pathway preferentially used by low-dose endotoxin in initiating low-grade inflammation.

The role of intestinal endotoxin in liver injury: a long and evolving history.

From the mid-1950s, it was observed that liver injury by a variety of toxins greatly sensitized the host to the effects of administered lipopolysaccharide. In the nutritional cirrhosis of choline deficiency, and in acute toxic injury as well, the need for the presence of enteric endotoxin was demonstrated. The universality of this association was striking for almost all agents associated with liver injury. In addition, the presence of endotoxemia in human liver disease was documented in the 1970s, when the hypothesis was first proposed, and correlated with the severity of the disease. Despite imposing evidence of the critical role of enteric endotoxin in liver injury, it did not excite much interest in investigators until the 1980s. With the ability to study effects of alcohol in newer delivery systems, and an increased understanding of the role of Kupffer cells in the process, the original hypothesis has been accepted. This historical review details the progress of this novel concept of disease initiation and suggests future directions to bring potential therapies to the bedside.

NADPH oxidase NOX2 mediates rapid cellular oxidation following ATP stimulation of endotoxin-primed macrophages.

The phagocytic NADPH oxidase (NOX2) plays a fundamental role in host defense and innate immunity. Here we demonstrate that external ATP triggers rapid cellular oxidation inhibited by diphenyleneiodonium in endotoxin-primed J774 macrophages and primary murine bone marrow-derived macrophages. To identify the source of reactive oxygen species (ROS), we compared responses between wild-type and NOX2-deficient macrophages. ATP-mediated ROS production was strongly attenuated in NOX2-deficient macrophages where responses were comparable to inhibition with diphenyleneiodonium. Notably, spatial differences in superoxide anion formation were observed where ROS formation was partially antagonized by extracellular superoxide dismutase in primary bone marrow-derived macrophages but unaffected in J774 macrophages. Loss of NOX2 was not observed to affect ATP-induced cell death. However, ATP-evoked cell death was found to be partially dependent on caspase-1 and cathepsin B activation. In conclusion, NOX2 plays a fundamental role in conferring macrophages with the ability to respond to extracellular ATP stimulation with robust changes in cellular oxidation.

Carboxy-terminal extension effects on crystal formation and insecticidal properties of Cry15Aa.

Cry15Aa protein, produced by Bacillus thuringiensis serovar thompsoni HD542, in a crystal together with a 40 kDa accompanying protein, is one of a small group of non-typical, less well-studied members of the Cry family of insecticidal proteins, and may provide an alternative for the more commonly used Cry proteins in insect pest management. In this study we examined the role of the C-terminal part of Cry15Aa and of the 40 kDa protein in crystal formation in recombinant B. thuringiensis. The contribution of the 40 kDa protein and of the Cry15Aa carboxy-terminal sequence for crystal formation, crystal solubilization, and insecticidal properties was assessed. No significant differences in toxicity against Cydia pomonella, before or after in vitro solubilization of crystal-spore preparations, were found. Although the 40 kDa protein significantly contributes to in vitro solubility and in vivo crystal formation of Cry15Aa, no direct evidence for involvement of the 40 kDa protein in toxicity of Cry15Aa was found.

Effect of heat stress on endotoxin flux across mesenteric-drained and portal-drained viscera of dairy goat.

This study was designed to evaluate the effect of heat stress on endotoxin flux across mesenteric-drained and portal-drained viscera of dairy goats. Three Saanen first lactation dairy goats were surgically fitted with indwelling catheters in the portal vein, the mesenteric vein and carotid, and were kept in thermal-neutral and then heat stress environment, for examining the effect of heat stress on endotoxin absorption and redox status. Average net absorption of endotoxin (EU/h) across mesenteric-drained viscera (MDV) and portal-drained viscera (PDV) during the whole period of heat stress increased by 279.05% and 227.92% in relation to thermo-neutral period. Plasma concentration of glutathione peroxidase (GSH-Px) and catalase (CAT) in mesenteric and portal vein, and that of superoxide dismutase (SOD) in mesenteric vein, increased significantly during heat stress. Main conclusions were: (i) net absorption of endotoxin in portal vein is mainly from non-mesenteric tissues both in heat stress and in thermo-neutral condition; (ii) heat stress may lead to the significant decrease in plasma SOD, GSH-Px, CAT flux across PDV and MDV, and the significant increase in endotoxin flux across PDV and MDV; and (iii) the increase in gastrointestinal permeability in dairy goats during heat stress may not be induced by the increase in oxidative stress.

Endotoxin in endodontic infections: a review.

Gram-negative bacteria play an essential role in primary endodontic infections. They have several virulence factors such as endotoxin, a large molecule that plays a role in the initiation and perpetuation of apical periodontitis. This paper reviews the role of gram-negative bacteria in endodontic infections, structure and mechanisms of action of endotoxin, endotoxin in infected root canals, effects of calcium hydroxide and polymixin B on endotoxin, and applications of endotoxin to measure leakage.

Bacillus thuringiensis Spores and Cry3A Toxins Act Synergistically to Expedite Colorado Potato Beetle Mortality.

The insect integument (exoskeleton) is an effective physiochemical barrier that limits disease-causing agents to a few portals of entry, including the gastrointestinal and reproductive tracts. The bacterial biopesticide Bacillus thuringiensis (Bt) enters the insect host via the mouth and must thwart gut-based defences to make its way into the body cavity (haemocoel) and establish infection. We sought to uncover the main antibacterial defences of the midgut and the pathophysiological features of Bt in a notable insect pest, the Colorado potato beetle Leptinotarsa decemlineata (CPB). Exposing the beetles to both Bt spores and their Cry3A toxins (crystalline δ-endotoxins) via oral inoculation led to higher mortality levels when compared to either spores or Cry3A toxins alone. Within 12 h post-exposure, Cry3A toxins caused a 1.5-fold increase in the levels of reactive oxygen species (ROS) and malondialdehyde (lipid peroxidation) within the midgut - key indicators of tissue damage. When Cry3A toxins are combined with spores, gross redox imbalance and 'oxidation stress' is apparent in beetle larvae. The insect detoxification system is activated when Bt spores and Cry3A toxins are administered alone or in combination to mitigate toxicosis, in addition to elevated mRNA levels of candidate defence genes (pattern-recognition receptor, stress-regulation, serine proteases, and prosaposin-like protein). The presence of bacterial spores and/or Cry3A toxins coincides with subtle changes in microbial community composition of the midgut, such as decreased Pseudomonas abundance at 48 h post inoculation. Both Bt spores and Cry3A toxins have negative impacts on larval health, and when combined, likely cause metabolic derangement, due to multiple tissue targets being compromised.

Effect of the hydrophobicity to net positive charge ratio on antibacterial and anti-endotoxin activities of structurally similar antimicrobial peptides.

The interaction between host-defense antimicrobial peptides (AMPs) and the bacterial lipopolysaccharide (LPS) governs both the susceptibility of the bacteria to the peptide and the ability of the peptide to inhibit LPS activation of immune cells. Both functions depend on the biophysical properties of the peptides. However, the sequence and structural diversity of AMPs makes it difficult to determine common denominators required for antimicrobial and LPS neutralizing activities. Toward this end, we synthesized and investigated a series of nine 12-amino acid peptides and their fatty acid-conjugated analogues composed of both D- and L-isomers of Leu and Lys at various ratios. The positions of the D-amino acids were preserved. These peptides differ in their net positive charge and hydrophobicity. However, their overall structure in the membrane is similar, as determined by Fourier transform infrared spectroscopy. The peptides and their analogues were functionally tested for their antibacterial and hemolytic activity, their ability to permeate LPS vesicles, their ability to neutralize LPS activation of macrophages, and their effect on LPS morphology, determined by negative staining electron microscopy. The data revealed that increasing the ratio between hydrophobicity and the net positive charge increases both antimicrobial and LPS neutralization activities, but with different modes of contributions. Whereas antimicrobial activity increases linearly with the increase in the peptides' hydrophobicity, peptides with different hydrophobicities are endowed with similar LPS neutralizing activities. Besides adding important information regarding AMP parameters involved in antimicrobial and anti-LPS activities, this study suggests the use of such diastereomers as potential templates for the development of simple molecules that conduct both types of functions.

A novel model of common Toll-like receptor 4- and injury-induced transcriptional themes in human leukocytes.

INTRODUCTION: An endotoxin challenge, sepsis, and injury/trauma, trigger significant changes in human peripheral blood leukocytes (PBL) gene expression. In this study, we have sought to test the hypothesis that the Toll-like receptor 4 (TLR4) induced transcription patterns elicited in humans exposed to in vivo endotoxin would parallel gene expression patterns observed in trauma patients with initial non-infectious injury. In addition, we sought to identify functional modules that are commonly affected by these two insults of differing magnitude and duration. METHODS: PBL were obtained from seven adult human subject experimental groups. The groups included a group of healthy, hospitalized volunteers (n = 15), that comprised four study groups of subjects challenged with intravenous endotoxin, without or with cortisol, and two serial samplings of trauma patients (n = 5). The PBL were analyzed for gene expression using a 8,793 probe microarray platform (Gene Chip® Focus, Affymetrix). The expression of a subset of genes was determined using qPCR. RESULTS: We describe sequential selection criteria of gene expression data that identifies 445 genes that are significantly differentially expressed (both P ≤ 0.05 and > 1.2 fold-change) in PBL derived from human subjects during the peak of systemic inflammatory responses induced by in vivo endotoxin, as well as in PBL obtained from trauma patients at 1 to 12 days after admission. We identified two functional modules that are commonly represented by this analysis. The first module includes more than 50 suppressed genes that encode ribosomal proteins or translation regulators. The second module includes up-regulated genes encoding key enzymes associated with glycolysis. Finally, we show that several circadian clock genes are also suppressed in PBL of surgical ICU patients. CONCLUSIONS: We identified a group of > 400 genes that exhibit similar expression trends in PBL derived from either endotoxin-challenged subjects or trauma patients. The suppressed translational and circadian clock modules, and the upregulated glycolytic module, constitute a robust and long lasting PBL gene expression signature that may provide a tool for monitoring systemic inflammation and injury.

Signaling versus punching hole: How do Bacillus thuringiensis toxins kill insect midgut cells?

Cry proteins, produced by Bacillus thuringiensis (Bt), are widely used for the control of insect pests in agriculture as spray products or expressed in transgenic crops, such as maize and cotton. Little was known regarding the mechanism of action of these toxins when the first commercial Bt product was introduced fifty years ago. However, research on the mechanism of action over the last two decades has enhanced our knowledge of toxin interaction with membrane receptors and their effects in insect midgut cells. All this information allowed for the rational design of improved toxins with higher toxicity or toxins that overcome insect resistance, which could compromise Bt use and effectiveness in the field. In this review we discuss and evaluate the different models of the mode of action of Cry toxins, including a discussion about the role of various receptors in toxin action.

[Myocardial dysfunction in sepsis--definition and pathogenetic mechanisms]. / Myokardiální dysfunkce v sepsi--charakteristika a patogenetické mechanizmy.

Sepsis is considered to be the major cause of morbidity and mortality of patients hospitalised in intensive care. It's defined as a systemic inflammatory response of organism to infection. Incidence of myocardial dysfunction in studies with severe sepsis patients is up to two thirds of patients. Cardiac dysfunction shows a continuum from isolated and mild diastolic dysfunction to combined severe diastolic and systolic failure of both ventricles mimicking even cardiogenic shock in some patients. Typical features of septic myocardial dysfunction (SMD) are decrease in ejection fraction (EF) with dilatation ofventricles, e.g. increase in end-diastolic volume (EDV). Reversibility of myocardial dysfunction during a period from 7 to 10 days in survivors is other typical manifestation of SMD. Hence, one can speculate that development of such a type ofSMD as a temporary protective compensatory mechanism could be advantageous for of an individual patient. A large body ofevidence about mechanisms ofSMD was described; endothelial dysfunction with consequent microcirculatory and mitochondrial dysfunction and role of circulating factors are considered to be the most important.

Chloroplast-targeted expression of synthetic cry1Ac in transgenic rice as an alternative strategy for increased pest protection.

To increase insect resistance in transgenic rice plants, a synthetic truncated cry1Ac gene was linked to the rice rbcS promoter and its transit peptide sequence (tp) for chloroplast-targeted expression. Several transgenic lines were generated by the Agrobacterium-mediated transformation method and the expression levels of the transgene were compared with untargeted expression. Use of the rbcS-tp sequence increased the cry1Ac transcript and protein levels by 25- and 100-fold, respectively, with the accumulated protein in chloroplasts comprising up to 2% of the total soluble proteins. The high level of cry1Ac expression resulted in high levels of plant resistance to three common rice pests, rice leaf folder, rice green caterpillar, and rice skipper, as evidenced by insect feeding assays. Transgenic plants were also evaluated for resistance to natural infestations by rice leaf folder under field conditions. Throughout the entire period of plant growth, the transgenic plants showed no symptoms of damage, whereas nontransgenic control plants were severely damaged by rice leaf folders. Our results demonstrate that the targeting of cry1Ac protein to the chloroplast using the rbcS:tp system confers a high level of plant protection to insects, thus providing an alternative strategy for crop insect management.

Endotoxin: stimulation of bone resorption in tissue culture.

Bacterial endotoxins can stimulate the release of previously incorporated calcium-45 and tritiated proline from fetal rat bone in tissue culture. Endotoxin from Bacteroides melaninogenicus, an organism regularly found in the gingival crevice of man, produces a response similar to parathyroid hormone and is effective at doses as low as 0.1 microgram per milliliter. This response is inhibited by serum and dependent upon the presence of albumin. Endotoxins may play a role in the bone loss characteristic of human periodontal disease.

N546 in beta18-beta19 loop is important for binding and toxicity of the Bacillus thuringiensis Cry1Ac toxin.

Our previous mutagenic analysis showed that the unique residue N546 in the apex of beta18-beta19 loop of Bacillus thuringiensis Cry1Ac toxin is important for its toxicity. In this study, trypsin digestion susceptibility, binding to BBMV and oligomer formation activity was therefore analyzed to determine the mechanism of toxicity change of these mutant toxins. The results showed that residue N546 was not involved in toxin oligomerisation and maintaining the stability of toxin, the enhanced toxicity of mutant N546A was just because of increased binding to BBMV, and reduction in toxicity of other mutants were caused by reduction in initial or irreversible binding to BBMV. This is the first report that revealed N546 in Cry1Ac domain III played an essential role in its insecticidal activity and binding to insect BBMV.

Assessment of biological and biochemical indicators in soil under transgenic Bt and non-Bt cotton crop in a sub-tropical environment.

There is concern that transgenic Bt-crops carry genes that could have undesirable effects on natural and agro-ecosystem functions. We investigated the effect of Bt-cotton (expressing the Cry 1Ac protein) on several microbial and biochemical indicators in a sandy loam soil. Bt-cotton (MRC-6301Bt) and its non-transgenic near-isoline (MRC-6301) were grown in a net-house on a sandy clay loam soil. Soil and root samples were collected 60, 90, and 120 days after sowing. Soil from a control (no-crop) treatment was also included. Samples were analysed for microbial biomass C, N and P (MBC, MBN, MBP), total organic carbon (TOC), and several soil enzyme activities. The microbial quotient (MQ) was calculated as the ratio of MBC-to-TOC. The average of the three sampling events revealed a significant increase in MBC, MBN, MBP and MQ in the soil under Bt-cotton over the non-Bt isoline. The TOC was similar in Bt and non-Bt systems. Potential N mineralization, nitrification, nitrate reductase, and acid and alkaline phosphatase activities were all higher in the soil under Bt-cotton. Root dry weights were not different (P > 0.05), but root volume of Bt-cotton was higher on 90 and 120 days than that of non-Bt cotton. The time of sampling strongly affected the above parameters, with most being highest on 90 days after sowing. We concluded from the data that there were some positive or no negative effects of Bt-cotton on the studied indicators, and therefore cultivation of Bt-cotton appears to be no risk to soil ecosystem functions.