Effect of gonococcal lipooligosaccharide variation on human monocytic cytokine profile

BACKGROUND: Neisseria gonorrhoeae is an obligate human pathogen that causes significant worldwide morbidity. N. gonorrhoeae expresses lipooligosaccharide (LOS), a phase variable molecule that plays an important role during pathogenesis of the organism. Alteration in the structure of gonococcal LOS correlates with altered disease presentation. In addition, LOS sialylation occurs readily in vivo, though the role of this sialylation during disease is unknown. RESULTS: Challenge of human monocytes with purified LOS preparations isolated from strains expressing distinct structurally defined LOSs resulted in identical production of the proinflammatory cytokines tumor necrosis factor alpha (TNFα) and interleukin-12 (IL-12). Similar results were seen when monocytes were challenged with either live or gentamicin-killed whole cell gonococcal variants expressing these LOS structures, although greater cytokine production was observed in comparison with challenge by purified LOS. Challenge of a human primary monocyte model with distinct LOS variants resulted in similar production of TNFα, IL-12, interleukin-10 (IL-10), and interleukin-8 (IL-8). A cytokine array was employed to allow measurement of a broad range of cytokines in samples challenge with gonococcal LOS variants as well as variants expressing sialylated LOS. Challenge of primary monocytes with sialylated gonococci was shown to elicit the production of more MCP-2 (monocyte chemoattractant protein-2) in comparison with challenge by unsialylated gonococci. CONCLUSION: We demonstrated that while alterations in the carbohydrate moiety of LOS do not impact the production of most cytokines by human monocytes, whole-cell bacterial challenge is more stimulatory than challenge with purified LOS, implying that other gonococcal cell surface antigens are important for the elicitation of cytokines. Challenge with gonococci expressing sialylated LOS resulted in elicitation of more of the chemokine MCP-2 from challenged cells in comparison with gonococci expressing unsialylated LOS. As MCP-2 is an important chemoattractant, this indicates that in vivo sialylation may play an important role during the pathogenesis of N. gonorrhoeae

Use of nfsB, encoding nitroreductase, as a reporter gene to determine the mutational spectrum of spontaneous mutations in Neisseria gonorrhoeae

<p>Abstract</p> <p>Background</p> <p>Organisms that are sensitive to nitrofurantoin express a nitroreductase. Since bacterial resistance to this compound results primarily from mutations in the gene encoding nitroreductase, the resulting loss of function of nitroreductase results in a selectable phenotype; resistance to nitrofurantoin. We exploited this direct selection for mutation to study the frequency at which spontaneous mutations arise (transitions and transversions, insertions and deletions).</p> <p>Results</p> <p>A nitroreductase- encoding gene was identified in the <it>N. gonorrhoeae </it>FA1090 genome by using a bioinformatic search with the deduced amino acid sequence derived from the <it>Escherichia coli </it>nitroreductase gene, <it>nfsB</it>. Cell extracts from <it>N. gonorrhoeae </it>were shown to possess nitroreductase activity, and activity was shown to be the result of NfsB. Spontaneous nitrofurantoin-resistant mutants arose at a frequency of ~3 × 10^-6- 8 × 10^-8among the various strains tested. The <it>nfsB </it>sequence was amplified from various nitrofurantoin-resistant mutants, and the nature of the mutations determined. Transition, transversion, insertion and deletion mutations were all readily detectable with this reporter gene.</p> <p>Conclusion</p> <p>We found that <it>nfsB </it>is a useful reporter gene for measuring spontaneous mutation frequencies. Furthermore, we found that mutations were more likely to arise in homopolymeric runs rather than as base substitutions.</p

Hysterosalpingography Versus Sonohysterography for Intrauterine Abnormalities

Sonohysterography was found to be more sensitive, specific, and accurate in the evaluation of specific intrauterine defects than hysterosalpingography

Genome engineering of isogenic human ES cells to model autism disorders.

Isogenic pluripotent stem cells are critical tools for studying human neurological diseases by allowing one to study the effects of a mutation in a fixed genetic background. Of particular interest are the spectrum of autism disorders, some of which are monogenic such as Timothy syndrome (TS); others are multigenic such as the microdeletion and microduplication syndromes of the 16p11.2 chromosomal locus. Here, we report engineered human embryonic stem cell (hESC) lines for modeling these two disorders using locus-specific endonucleases to increase the efficiency of homology-directed repair (HDR). We developed a system to: (1) computationally identify unique transcription activator-like effector nuclease (TALEN) binding sites in the genome using a new software program, TALENSeek, (2) assemble the TALEN genes by combining golden gate cloning with modified constructs from the FLASH protocol, and (3) test the TALEN pairs in an amplification-based HDR assay that is more sensitive than the typical non-homologous end joining assay. We applied these methods to identify, construct, and test TALENs that were used with HDR donors in hESCs to generate an isogenic TS cell line in a scarless manner and to model the 16p11.2 copy number disorder without modifying genomic loci with high sequence similarity

Characterization of the dsDNA prophage sequences in the genome of Neisseria gonorrhoeae and visualization of productive bacteriophage

<p>Abstract</p> <p>Background</p> <p>Bioinformatic analysis of the genome sequence of <it>Neisseria gonorrhoeae </it>revealed the presence of nine probable prophage islands. The distribution, conservation and function of many of these sequences, and their ability to produce bacteriophage particles are unknown.</p> <p>Results</p> <p>Our analysis of the genomic sequence of FA1090 identified five genomic regions (NgoΦ1 – 5) that are related to dsDNA lysogenic phage. The genetic content of the dsDNA prophage sequences were examined in detail and found to contain blocks of genes encoding for proteins homologous to proteins responsible for phage DNA replication, structural proteins and proteins responsible for phage assembly. The DNA sequences from NgoΦ1, NgoΦ2 and NgoΦ3 contain some significant regions of identity. A unique region of NgoΦ2 showed very high similarity with the <it>Pseudomonas aeruginosa </it>generalized transducing phage F116. Comparative analysis at the nucleotide and protein levels suggests that the sequences of NgoΦ1 and NgoΦ2 encode functionally active phages, while NgoΦ3, NgoΦ4 and NgoΦ5 encode incomplete genomes. Expression of the NgoΦ1 and NgoΦ2 repressors in <it>Escherichia coli </it>inhibit the growth of <it>E. coli </it>and the propagation of phage λ. The NgoΦ2 repressor was able to inhibit transcription of <it>N. gonorrhoeae </it>genes and <it>Haemophilus influenzae </it>HP1 phage promoters. The holin gene of NgoΦ1 (identical to that encoded by NgoΦ2), when expressed in <it>E. coli</it>, could serve as substitute for the phage λ <it>s </it>gene. We were able to detect the presence of the DNA derived from NgoΦ1 in the cultures of <it>N. gonorrhoeae</it>. Electron microscopy analysis of culture supernatants revealed the presence of multiple forms of bacteriophage particles.</p> <p>Conclusion</p> <p>These data suggest that the genes similar to dsDNA lysogenic phage present in the gonococcus are generally conserved in this pathogen and that they are able to regulate the expression of other neisserial genes. Since phage particles were only present in culture supernatants after induction with mitomycin C, it indicates that the gonococcus also regulates the expression of bacteriophage genes.</p

Preventing problematic internet use during the COVID-19 pandemic: Consensus guidance

As a response to the COVID-19 pandemic, many governments have introduced steps such as spatial distancing and "staying at home" to curb its spread and impact. The fear resulting from the disease, the lockdown' situation, high levels of uncertainty regarding the future, and financial insecurity raise the level of stress, anxiety, and depression experienced by people all around the world. Psychoactive substances and other reinforcing behaviors (e.g., gambling, video gaming, watching pornography) are often used to reduce stress and anxiety and/or to alleviate depressed mood. The tendency to use such substances and engage in such behaviors in an excessive manner as putative coping strategies in crises like the COVID-19 pandemic is considerable. Moreover, the importance of information and communications technology (ICT) is even higher in the present crisis than usual. ICT has been crudal in keeping parts of the economy going, allowing large groups of people to work and study from home, enhancing social connectedness, providing greatly needed entertainment, etc. Although for the vast majority ICT use is adaptive and should not be pathologized, a subgroup of vulnerable individuals are at risk of developing problematic usage patterns. The present consensus guidance discusses these risks and makes some practical recommendations that may help diminish them

Size-dependent rheology of type-I collagen networks

We investigate the system size dependent rheological response of branched type I collagen gels. When subjected to a shear strain, the highly interconnected mesh dynamically reorients, resulting in overall stiffening of the network. When a continuous shear strain is applied to a collagen network, we observe that the local apparent modulus, in the strain-stiffening regime, is strongly dependent on the gel thickness. In addition, we demonstrate that the overall network failure is determined by the ratio of the gel thickness to the mesh size. These findings have broad implications for cell-matrix interactions, the interpretation of rheological tissue data, and the engineering of biomimetic scaffolds.Comment: 3 pages, 4 figures, to appear in Biophysical Journal Letters, September 201