The molecular characterisation of Escherichia coli K1 isolated from neonatal nasogastric feeding tubes

Background: The most common cause of Gram-negative bacterial neonatal meningitis is E. coli K1. It has a mortality rate of 10–15%, and neurological sequelae in 30– 50% of cases. Infections can be attributable to nosocomial sources, however the pre-colonisation of enteral feeding tubes has not been considered as a specific risk factor. Methods: Thirty E. coli strains, which had been isolated in an earlier study, from the residual lumen liquid and biofilms of neonatal nasogastric feeding tubes were genotyped using pulsed-field gel electrophoresis, and 7-loci multilocus sequence typing. Potential pathogenicity and biofilm associated traits were determined using specific PCR probes, genome analysis, and in vitro tissue culture assays. Results: The E. coli strains clustered into five pulsotypes, which were genotyped as sequence types (ST) 95, 73, 127, 394 and 2076 (Achman scheme). The extra-intestinal pathogenic E. coli (ExPEC) phylogenetic group B2 ST95 serotype O1:K1:NM strains had been isolated over a 2 week period from 11 neonates who were on different feeding regimes. The E. coli K1 ST95 strains encoded for various virulence traits associated with neonatal meningitis and extracellular matrix formation. These strains attached and invaded intestinal, and both human and rat brain cell lines, and persisted for 48 h in U937 macrophages. E. coli STs 73, 394 and 2076 also persisted in macrophages and invaded Caco-2 and human brain cells, but only ST394 invaded rat brain cells. E. coli ST127 was notable as it did not invade any cell lines. Conclusions: Routes by which E. coli K1 can be disseminated within a neonatal intensive care unit are uncertain, however the colonisation of neonatal enteral feeding tubes may be one reservoir source which could constitute a serious health risk to neonates following ingestion

Neuroinflammation, Mast Cells, and Glia: Dangerous Liaisons

The perspective of neuroinflammation as an epiphenomenon following neuron damage is being replaced by the awareness of glia and their importance in neural functions and disorders. Systemic inflammation generates signals that communicate with the brain and leads to changes in metabolism and behavior, with microglia assuming a pro-inflammatory phenotype. Identification of potential peripheral-to-central cellular links is thus a critical step in designing effective therapeutics. Mast cells may fulfill such a role. These resident immune cells are found close to and within peripheral nerves and in brain parenchyma/meninges, where they exercise a key role in orchestrating the inflammatory process from initiation through chronic activation. Mast cells and glia engage in crosstalk that contributes to accelerate disease progression; such interactions become exaggerated with aging and increased cell sensitivity to stress. Emerging evidence for oligodendrocytes, independent of myelin and support of axonal integrity, points to their having strong immune functions, innate immune receptor expression, and production/response to chemokines and cytokines that modulate immune responses in the central nervous system while engaging in crosstalk with microglia and astrocytes. In this review, we summarize the findings related to our understanding of the biology and cellular signaling mechanisms of neuroinflammation, with emphasis on mast cell-glia interactions

Trypanosoma brucei PUF9 Regulates mRNAs for Proteins Involved in Replicative Processes over the Cell Cycle

Many genes that are required at specific points in the cell cycle exhibit cell cycle–dependent expression. In the early-diverging model eukaryote and important human pathogen Trypanosoma brucei, regulation of gene expression in the cell cycle and other processes is almost entirely post-transcriptional. Here, we show that the T. brucei RNA-binding protein PUF9 stabilizes certain transcripts during S-phase. Target transcripts of PUF9—LIGKA, PNT1 and PNT2—were identified by affinity purification with TAP-tagged PUF9. RNAi against PUF9 caused an accumulation of cells in G2/M phase and unexpectedly destabilized the PUF9 target mRNAs, despite the fact that most known Puf-domain proteins promote degradation of their target mRNAs. The levels of the PUF9-regulated transcripts were cell cycle dependent, peaking in mid- to late- S-phase, and this effect was abolished when PUF9 was targeted by RNAi. The sequence UUGUACC was over-represented in the 3′ UTRs of PUF9 targets; a point mutation in this motif abolished PUF9-dependent stabilization of a reporter transcript carrying the PNT1 3′ UTR. LIGKA is involved in replication of the kinetoplast, and here we show that PNT1 is also kinetoplast-associated and its over-expression causes kinetoplast-related defects, while PNT2 is localized to the nucleus in G1 phase and redistributes to the mitotic spindle during mitosis. PUF9 targets may constitute a post-transcriptional regulon, encoding proteins involved in temporally coordinated replicative processes in early G2 phase

GPER mediates the angiocrine actions induced by IGF1 through the HIF-1α/VEGF pathway in the breast tumor microenvironment

The G protein estrogen receptor GPER/GPR30 mediates estrogen action in breast cancer cells as well as in breast cancer-associated fibroblasts (CAFs), which are key components of microenvironment driving tumor progression. GPER is a transcriptional target of hypoxia inducible factor 1 alpha (HIF-1α) and activates VEGF expression and angiogenesis in hypoxic breast tumor microenvironment. Furthermore, IGF1/IGF1R signaling, which has angiogenic effects, has been shown to activate GPER in breast cancer cells. We analyzed gene expression data from published studies representing almost 5000 breast cancer patients to investigate whether GPER and IGF1 signaling establish an angiocrine gene signature in breast cancer patients. Next, we used GPER-positive but estrogen receptor (ER)-negative primary CAF cells derived from patient breast tumours and SKBR3 breast cancer cells to investigate the role of GPER in the regulation of VEGF expression and angiogenesis triggered by IGF1. We performed gene expression and promoter studies, western blotting and immunofluorescence analysis, gene silencing strategies and endothelial tube formation assays to evaluate the involvement of the HIF-1α/GPER/VEGF signaling in the biological responses to IGF1. We first determined that GPER is co-expressed with IGF1R and with the vessel marker CD34 in human breast tumors (n = 4972). Next, we determined that IGF1/IGF1R signaling engages the ERK1/2 and AKT transduction pathways to induce the expression of HIF-1α and its targets GPER and VEGF. We found that a functional cooperation between HIF-1α and GPER is essential for the transcriptional activation of VEGF induced by IGF1. Finally, using conditioned medium from CAFs and SKBR3 cells stimulated with IGF1, we established that HIF-1α and GPER are both required for VEGF-induced human vascular endothelial cell tube formation. These findings shed new light on the essential role played by GPER in IGF1/IGF1R signaling that induces breast tumor angiogenesis. Targeting the multifaceted interactions between cancer cells and tumor microenvironment involving both GPCRs and growth factor receptors has potential in future combination anticancer therapies

Blockade of VEGFR1 and 2 Suppresses Pathological Angiogenesis and Vascular Leakage in the Eye

VEGFR1 and 2 signaling have both been increasingly shown to mediate complications of ischemic retinopathies, including retinopathy of prematurity (ROP), age-related macular degeneration (AMD), and diabetic retinopathy (DR). This study evaluates the effects of blocking VEGFR1 and 2 on pathological angiogenesis and vascular leakage in ischemic retinopathy in a model of ROP and in choroidal neovascularization (CNV) in a model of AMD.H]-mannitol leakage from blood vessels into the retina. Gene expression was measured by real-time quantitative (Q)PCR.VEGFR1 and VEGFR2 expressions were up-regulated during CNV pathogenesis. Both MF1 and DC101 significantly suppressed CNV at 50 mg/kg: DC101 suppressed CNV by 73±5% (p<0.0001) and MF1 by 64±6% (p = 0.0002) in a dosage-dependent manner. The combination of MF1 and DC101 enhanced the inhibitory efficacy and resulted in an accumulation of retinal microglia at the CNV lesion. Similarly, both MF1 and DC101 significantly suppressed retinal NV in OIR at 50 mg/kg: DC101 suppressed retinal NV by 54±8% (p = 0.013) and MF1 by 50±7% (p<0.0002). MF1 was even more effective at inhibiting ischemia-induced BRB breakdown than DC101: the retina/lung leakage ratio for MF1 was reduced by 73±24%, p = 0.001 and for DC101 by 12±4%, p = 0.003. The retina/renal leakage ratio for MF1 was reduced by 52±28%, p = 0.009 and for DC101 by 13±4%, p = 0.001.Our study provides further evidence that both VEGFR1 and 2 mediate pathological angiogenesis and vascular leakage in these models of ocular disease and suggests that antagonist antibodies to these receptor tyrosine kinases (RTKs) are potential therapeutic agents

Novel Structural Components of the Ventral Disc and Lateral Crest in Giardia intestinalis

Giardia intestinalis is a ubiquitous parasitic protist that is the causative agent of giardiasis, one of the most common protozoan diarrheal diseases in the world. Giardia trophozoites attach to the intestinal epithelium using a specialized and elaborate microtubule structure, the ventral disc. Surrounding the ventral disc is a less characterized putatively contractile structure, the lateral crest, which forms a continuous perimeter seal with the substrate. A better understanding of ventral disc and lateral crest structure, conformational dynamics, and biogenesis is critical for understanding the mechanism of giardial attachment to the host. To determine the components comprising the ventral disc and lateral crest, we used shotgun proteomics to identify proteins in a preparation of isolated ventral discs. Candidate disc-associated proteins, or DAPs, were GFP-tagged using a ligation-independent high-throughput cloning method. Based on disc localization, we identified eighteen novel DAPs, which more than doubles the number of known disc-associated proteins. Ten of the novel DAPs are associated with the lateral crest or outer edge of the disc, and are the first confirmed components of this structure. Using Fluorescence Recovery After Photobleaching (FRAP) with representative novel DAP::GFP strains we found that the newly identified DAPs tested did not recover after photobleaching and are therefore structural components of the ventral disc or lateral crest. Functional analyses of the novel DAPs will be central toward understanding the mechanism of ventral disc-mediated attachment and the mechanism of disc biogenesis during cell division. Since attachment of Giardia to the intestine via the ventral disc is essential for pathogenesis, it is possible that some proteins comprising the disc could be potential drug targets if their loss or disruption interfered with disc biogenesis or function, preventing attachment

Aging of the mammalian gastrointestinal tract: a complex organ system

Gastrointestinal disorders are a major cause of morbidity in the elderly population. The gastrointestinal tract is the most complex organ system; its diverse cells perform a range of functions essential to life, not only secretion, digestion, absorption and excretion, but also, very importantly, defence. The gastrointestinal tract acts not only as a barrier to harmful materials and pathogens but also contains the vast number of beneficial bacterial populations that make up the microbiota. Communication between the cells of the gastrointestinal tract and the central nervous and endocrine systems modifies behaviour; the organisms of the microbiota also contribute to this brain–gut–enteric microbiota axis. Age-related physiological changes in the gut are not only common, but also variable, and likely to be influenced by external factors as well as intrinsic aging of the cells involved. The cellular and molecular changes exhibited by the aging gut cells also vary. Aging intestinal smooth muscle cells exhibit a number of changes in the signalling pathways that regulate contraction. There is some evidence for age-associated degeneration of neurons and glia of the enteric nervous system, although enteric neuronal losses are likely not to be nearly as extensive as previously believed. Aging enteric neurons have been shown to exhibit a senescence-associated phenotype. Epithelial stem cells exhibit increased mitochondrial mutation in aging that affects their progeny in the mucosal epithelium. Changes to the microbiota and intestinal immune system during aging are likely to contribute to wider aging of the organism and are increasingly important areas of analysis. How changes of the different cell types of the gut during aging affect the numerous cellular interactions that are essential for normal gut functions will be important areas for future aging research

Association of insularity and body condition to cloacal bacteria prevalence in a small shorebird

Do islands harbour less diverse disease communities than mainland? The island biogeography theory predicts more diverse communities on mainland than on islands due to more niches, more diverse habitats and availability of greater range of hosts. We compared bacteria prevalences ofCampylobacter,ChlamydiaandSalmonellain cloacal samples of a small shorebird, the Kentish plover (Charadrius alexandrinus) between two island populations of Macaronesia and two mainland locations in the Iberian Peninsula. Bacteria were found in all populations but, contrary to the expectations, prevalences did not differ between islands and mainland. Females had higher prevalences than males forSalmonellaand when three bacteria genera were pooled together. Bacteria infection was unrelated to bird's body condition but females from mainland were heavier than males and birds from mainland were heavier than those from islands. Abiotic variables consistent throughout breeding sites, like high salinity that is known to inhibit bacteria growth, could explain the lack of differences in the bacteria prevalence between areas. We argue about the possible drivers and implications of sex differences in bacteria prevalence in Kentish plovers