Known pathogenic gene variants and new candidates detected in Sudden Unexpected Infant Death using Whole Genome Sequencing.

Purpose: To gain insights into potential genetic factors contributing to the infant's vulnerability to Sudden Unexpected Infant Death (SUID). Methods: Whole Genome Sequencing (WGS) was performed on 145 infants that succumbed to SUID, and 576 healthy adults. Variants were filtered by gnomAD allele frequencies and predictions of functional consequences. Results: Variants of interest were identified in 86 genes, 63.4% of our cohort. Seventy-one of these have been previously associated with SIDS/SUID/SUDP. Forty-three can be characterized as cardiac genes and are related to cardiomyopathies, arrhythmias, and other conditions. Variants in 22 genes were associated with neurologic functions. Variants were also found in 13 genes reported to be pathogenic for various systemic disorders. Variants in eight genes are implicated in the response to hypoxia and the regulation of reactive oxygen species (ROS) and have not been previously described in SIDS/SUID/SUDP. Seventy-two infants met the triple risk hypothesis criteria (Figure 1). Conclusion: Our study confirms and further expands the list of genetic variants associated with SUID. The abundance of genes associated with heart disease and the discovery of variants associated with the redox metabolism have important mechanistic implications for the pathophysiology of SUID.

Studies on the anti-diarrheal properties of leaf extract of Desmodium puchellum.

OBJECTIVE: To evaluate the pharmacological activity against diarrhea of methanol and petroleum ether extract of Desmodium puchellum (Family: Fabaceae) leaves. METHODS: The extract was evaluated for castor oil-induced diarrhea and enteropooling as well as intestinal motility in rats. Both of the extracts were given to the rats at 200 mg/kg orally. Loperamide was used as a standard drug for diarrhea. RESULTS: The diarrheal severity was reduced significantly (P<0.05) by methanol and petroleum ether extracts by 31.95% and 28.33%, respectively, whereas 54.75% inhibition was found for standard drug loperamide at 5 mg/kg. The two extracts also significantly (P<0.05) reduced the intestinal volume in case of castor oil induced enteropooling. CONCLUSIONS: It is concluded that both fractions contain some biologically active ingredients that are active for anti-diarrheal actions whereas methanol fraction has better potential.

Toxin inhibition of antimicrobial factors induced by Bacillus anthracis peptidoglycan in human blood.

Here, we describe the capacity of Bacillus anthracis peptidoglycan (BaPGN) to trigger an antimicrobial response in human white blood cells (WBCs). Analysis of freshly isolated human blood cells found that monocytes and neutrophils, but not B and T cells, were highly responsive to BaPGN and produced a variety of cytokines and chemokines. This BaPGN-induced response was suppressed by anthrax lethal toxin (LT) and edema toxin (ET), with the most pronounced effect on human monocytes, and this corresponded with the higher levels of anthrax toxin receptor 1 (ANTXR1) in these cells than in neutrophils. The supernatant from BaPGN-treated cells altered the growth of B. anthracis Sterne, and this effect was blocked by LT, but not by ET. An FtsX mutant of B. anthracis known to be resistant to the antimicrobial effects of interferon-inducible Glu-Leu-Arg (ELR)-negative CXC chemokines was not affected by the BaPGN-induced antimicrobial effects. Collectively, these findings describe a system in which BaPGN triggers expression of antimicrobial factors in human WBCs and reveal a distinctive role, not shared with ET, in LT's capacity to suppress this response.

Increased cAMP in monocytes augments Notch signaling mechanisms by elevating RBP-J and transducin-like enhancer of Split (TLE).

In cells of the innate immune system, pathological increases in intracellular cAMP attenuate immune responses and contribute to infections by bacteria such as Bacillus anthracis. In this work, cAMP from B. anthracis edema toxin (ET) is found to activate the Notch signaling pathway in both mouse macrophages and human monocytes. ET as well as a cell-permeable activator of PKA induce Notch target genes (HES1, HEY1, IL2RA, and IL7R) and are able to significantly enhance the induction of these Notch target genes by a Toll-like receptor ligand. Elevated cAMP also resulted in increased levels of Groucho/transducin-like enhancer of Split (TLE) and led to increased amounts of a transcriptional repressor complex consisting of TLE and the Notch target Hes1. To address the mechanism used by ET to activate Notch signaling, components of Notch signaling were examined, and results revealed that ET increased levels of recombinant recognition sequence binding protein at the Jκ site (RBP-J), a DNA binding protein and principal transcriptional regulator of Notch signaling. Overexpression studies indicated that RBP-J was sufficient to activate Notch signaling and potentiate LPS-induced Notch signaling. Further examination of the mechanism used by ET to activate Notch signaling revealed that C/EBP ß, a transcription factor activated by cAMP, helped activate Notch signaling and up-regulated RBP-J. These studies demonstrate that cAMP activates Notch signaling and increases the expression of TLE, which could be an important mechanism utilized by cAMP to suppress immune responses.

Differential inflammatory responses triggered by toxic small molecules.

PURPOSE: The aim of this study is to determine whether exposure to hazardous chemicals alters chemokine or cytokine production in macrophages and link these events to changes in intracellular signaling pathways and activation of specific gene promoters. METHODS: RAW 264.7 mouse macrophages were treated with selected toxic industrial chemicals (TICs) and examined for changes in immune function. Luminex multiplex technology was used to assess changes in cytokine/chemokine expression and activation of kinase signaling pathways. In addition, a panel of macrophage cell lines with promoter-specific luciferase reporter genes were generated and treated with the TICs, and transcriptional responses to these chemicals were detected by changes in luminescence. RESULTS: Changes in expression of cytokines and chemokines were linked to changes in the activation state of intracellular signaling pathways. Overall, the findings reveal that sublytic levels of TICs can alter the profile of cytokines and chemokines expressed by macrophages, with a pattern that suggests immunosuppression. The data demonstrate that critical changes in immune function correlate with activation of kinase signaling pathways in macrophages. CONCLUSIONS: These data provide insight into the effects of sublytic doses of selected TICs on macrophage function, with a particular emphasis on identifying changes in expression of cytokines and chemokines. These altered patterns in immune function were linked to changes in the activation state of intracellular signaling pathways. The data strongly suggest that small amounts of TICs can have subtle, yet very critical, effects on macrophages.

Variations in TcdB activity and the hypervirulence of emerging strains of Clostridium difficile.

Hypervirulent strains of Clostridium difficile have emerged over the past decade, increasing the morbidity and mortality of patients infected by this opportunistic pathogen. Recent work suggested the major C. difficile virulence factor, TcdB, from hypervirulent strains (TcdB(HV)) was more cytotoxic in vitro than TcdB from historical strains (TcdB(HIST)). The current study investigated the in vivo impact of altered TcdB tropism, and the underlying mechanism responsible for the differences in activity between the two forms of this toxin. A combination of protein sequence analyses, in vivo studies using a Danio rerio model system, and cell entry combined with fluorescence assays were used to define the critical differences between TcdB(HV) and TcdB(HIST). Sequence analysis found that TcdB was the most variable protein expressed from the pathogenicity locus of C. difficile. In line with these sequence differences, the in vivo effects of TcdB(HV) were found to be substantially broader and more pronounced than those caused by TcdB(HIST). The increased toxicity of TcdB(HV) was related to the toxin's ability to enter cells more rapidly and at an earlier stage in endocytosis than TcdB(HIST). The underlying biochemical mechanism for more rapid cell entry was identified in experiments demonstrating that TcdB(HV) undergoes acid-induced conformational changes at a pH much higher than that of TcdB(HIST). Such pH-related conformational changes are known to be the inciting step in membrane insertion and translocation for TcdB. These data provide insight into a critical change in TcdB activity that contributes to the emerging hypervirulence of C. difficile.

Metagenomic insights into evolution of a heavy metal-contaminated groundwater microbial community.

Understanding adaptation of biological communities to environmental change is a central issue in ecology and evolution. Metagenomic analysis of a stressed groundwater microbial community reveals that prolonged exposure to high concentrations of heavy metals, nitric acid and organic solvents ( approximately 50 years) has resulted in a massive decrease in species and allelic diversity as well as a significant loss of metabolic diversity. Although the surviving microbial community possesses all metabolic pathways necessary for survival and growth in such an extreme environment, its structure is very simple, primarily composed of clonal denitrifying gamma- and beta-proteobacterial populations. The resulting community is overabundant in key genes conferring resistance to specific stresses including nitrate, heavy metals and acetone. Evolutionary analysis indicates that lateral gene transfer could have a key function in rapid response and adaptation to environmental contamination. The results presented in this study have important implications in understanding, assessing and predicting the impacts of human-induced activities on microbial communities ranging from human health to agriculture to environmental management, and their responses to environmental changes.

Impacts of Shewanella oneidensis c-type cytochromes on aerobic and anaerobic respiration.

Shewanella are renowned for their ability to utilize a wide range of electron acceptors (EA) for respiration, which has been partially accredited to the presence of a large number of the c-type cytochromes. To investigate the involvement of c-type cytochrome proteins in aerobic and anaerobic respiration of Shewanella oneidensis Mr -1, 36 in-frame deletion mutants, among possible 41 predicted, c-type cytochrome genes were obtained. The potential involvement of each individual c-type cytochrome in the reduction of a variety of EAs was assessed individually as well as in competition experiments. While results on the well-studied c-type cytochromes CymA(SO4591) and MtrC(SO1778) were consistent with previous findings, collective observations were very interesting: the responses of S. oneidensis Mr -1 to low and highly toxic metals appeared to be significantly different; CcoO, CcoP and PetC, proteins involved in aerobic respiration in various organisms, played critical roles in both aerobic and anaerobic respiration with highly toxic metals as EA. In addition, these studies also suggested that an uncharacterized c-type cytochrome (SO4047) may be important to both aerobiosis and anaerobiosis.

Reduction of nitrate in Shewanella oneidensis depends on atypical NAP and NRF systems with NapB as a preferred electron transport protein from CymA to NapA.

In the genome of Shewanella oneidensis, a napDAGHB gene cluster encoding periplasmic nitrate reductase (NapA) and accessory proteins and an nrfA gene encoding periplasmic nitrite reductase (NrfA) have been identified. These two systems seem to be atypical because the genome lacks genes encoding cytoplasmic membrane electron transport proteins, NapC for NAP and NrfBCD/NrfH for NRF, respectively. Here, we present evidence that reduction of nitrate to ammonium in S. oneidensis is carried out by these atypical systems in a two-step manner. Transcriptional and mutational analyses suggest that CymA, a cytoplasmic membrane electron transport protein, is likely to be the functional replacement of both NapC and NrfH in S. oneidensis. Surprisingly, a strain devoid of napB encoding the small subunit of nitrate reductase exhibited the maximum cell density sooner than the wild type. Further characterization of this strain showed that nitrite was not detected as a free intermediate in its culture and NapB provides a fitness gain for S. oneidensis to compete for nitrate in the environments. On the basis results from mutational analyses of napA, napB, nrfA and napBnrfA in-frame deletion mutants, we propose that NapB is able to favor nitrate reduction by routing electrons to NapA exclusively.

The mechanism of Bacillus anthracis intracellular germination requires multiple and highly diverse genetic loci.

In an effort to better understand the mechanisms by which Bacillus anthracis establishes disease, experiments were undertaken to identify the genes essential for intracellular germination. Eighteen diverse genetic loci were identified via an enrichment protocol using a transposon-mutated library of B. anthracis spores, which was screened for mutants delayed in intracellular germination. Fourteen transposon mutants were identified in genes not previously associated with B. anthracis germination and included disruption of factors involved in membrane transport, transcriptional regulation, and intracellular signaling. Four mutants contained transposon insertions in gerHA, gerHB, gerHC, and pagA, respectively, each of which has been previously associated with germination or survival of B. anthracis within macrophages. Strain MIGD101 (named for macrophage intracellular germination defective 101) was of particular interest, since this mutant contained a transposon insertion in an intergenic region between BAs2807 and BAs2808, and was the most highly represented mutant in the enrichment. Analysis of B. anthracis MIGD101 by confocal microscopy and differential heat sensitivity following macrophage infection revealed ungerminated spores within the cell. Moreover, B. anthracis MIGD101 was attenuated in cell killing relative to the parent strain. Further experimental analysis found that B. anthracis MIGD101 was defective in five known B. anthracis germination pathways, supporting a mechanism wherein the intergenic region between BAs2807 and BAs2808 has a global affect on germination of this pathogen. Collectively, these findings provide insight into the mechanisms supporting B. anthracis germination within host cells.

Current production and metal oxide reduction by Shewanella oneidensis MR-1 wild type and mutants.

Shewanella oneidensis MR-1 is a gram-negative facultative anaerobe capable of utilizing a broad range of electron acceptors, including several solid substrates. S. oneidensis MR-1 can reduce Mn(IV) and Fe(III) oxides and can produce current in microbial fuel cells. The mechanisms that are employed by S. oneidensis MR-1 to execute these processes have not yet been fully elucidated. Several different S. oneidensis MR-1 deletion mutants were generated and tested for current production and metal oxide reduction. The results showed that a few key cytochromes play a role in all of the processes but that their degrees of participation in each process are very different. Overall, these data suggest a very complex picture of electron transfer to solid and soluble substrates by S. oneidensis MR-1.

Involvement of surface polysaccharides in the organic acid resistance of Shiga Toxin-producing Escherichia coli O157:H7.

In general, wild Escherichia coli strains can grow effectively under moderately acidic organic acid-rich conditions. We found that the Shiga Toxin-producing E. coli (STEC) O157:H7 NGY9 grows more quickly than a K-12 strain in Luria-Bertani (LB)-2-morpholinoethanesulphonic acid (MES) broth supplemented with acetic acid (pH 5.4). Hypothesizing that the resistance of STEC O157:H7 to acetic acid is as a result of a mechanism(s) other than those known, we screened for STEC mutants sensitive to acetic acid. NGY9 was subjected to mini-Tn5 mutagenesis and, from 50,000 colonies, five mutants that showed a clear acetic acid-sensitive phenotype were isolated. The insertion of mini-Tn5 in three mutants occurred at the fcl, wecA (rfe) and wecB (rffE) genes and caused loss of surface O-polysaccharide, loss of both O-polysaccharide and enterobacterial common antigen (ECA) and loss of ECA respectively. The other two mutants showed inactivation of the waaG (rfaG) gene but at different positions that caused a deep rough mutant with loss of the outer core oligosaccharide of lipopolysaccharide (LPS) as well as phenotypic loss of O-polysaccharide and ECA. With the introduction of plasmids carrying the fcl, wecA, wecB and waaG genes, respectively, all mutants were complemented in their production of O-polysaccharide and ECA, and normal growth was restored in organic acid-rich culture conditions. We also found that the growth of Salmonella LPS mutants Ra, Rb1, Rc, Rd1, Rd2 and Re was suppressed in the presence of acetic acid compared with that of the parents. These results suggest that the full expression of LPS (including O-polysaccharide) and ECA is indispensable to the resistance against acetic acid and other short chain fatty acids in STEC O157:H7 and Salmonella. To the best of our knowledge, this is a newly identified physiological role for O-polysaccharide and ECA as well as an acid resistance mechanism.