Isolation of Phages for Phage Therapy: A Comparison of Spot Tests and Efficiency of Plating Analyses for Determination of Host Range and Efficacy

Phage therapy, treating bacterial infections with bacteriophages, could be a future alternative to antibiotic treatment of bacterial infections. There are, however, several problems to be solved, mainly associated to the biology of phages, the interaction between phages and their bacterial hosts, but also to the vast variation of pathogenic bacteria which implies that large numbers of different phages are going to be needed. All of these phages must under present regulation of medical products undergo extensive clinical testing before they can be applied. It will consequently be of great economic importance that effective and versatile phages are selected and collected into phage libraries, i.e., the selection must be carried out in a way that it results in highly virulent phages with broad host ranges. We have isolated phages using the Escherichia coli reference (ECOR) collection and compared two methods, spot testing and efficiency of plating (EOP), which are frequently used to identify phages suitable for phage therapy. The analyses of the differences between the two methods show that spot tests often overestimate both the overall virulence and the host range and that the results are not correlated to the results of EOP assays. The conclusion is that single dilution spot tests cannot be used for identification and selection of phages to a phage library and should be replaced by EOP assays. The difference between the two methods can be caused by many factors. We have analysed if the differences and lack of correlation could be caused by lysis from without, bacteriocins in the phage lysate, or by the presence of prophages harbouring genes coding for phage resistance systems in the genomes of the bacteria in the ECOR collection.     

The potential role of carbon dioxide in the neuroimmunoendocrine changes following cerebral ischemia

Carbon dioxide (CO(2)) interacts in complex ways with the brain and the endocrine and immune systems. Arterial CO(2) may be elevated or decreased following cerebral ischemia-reperfusion injury or stroke. The aim of the present review is to delineate potential changes in the neuroimmunoendocrine system following cerebral ischemia-reperfusion injury and to provide evidence for the modulatory role of carbon dioxide in this setting. It appears that lesions of the right and left cerebral hemispheres are associated with different patterns of immune activation and cytokine release. Changes in arterial CO(2) can profoundly alter the neuroimmunoendocrine system, especially the hypothalamic-pituitary-adrenal (HPA) axis and the production of pro-inflammatory cytokines. Hypercapnia activates the HPA axis, exerts antiinflammatory and antioxidant effects, and can alter the secretion and function of various brain neurotransmitters. There is conflicting evidence surrounding arterial CO(2): its effects on the ischemic brain may be either beneficial or deleterious. Mild hypercapnia may exert some neuroprotection following cerebral ischemia, but severe hypercapnia may aggravate neuronal injury by extra- and intra-cellular acidification and/or impairment of cellular calcium hemostasis. Future studies are required to delineate the potential relationship between arterial CO(2) and prognosis and long-term survival following cerebral ischemia-reperfusion injury. "Therapeutic hypercapnia" seems to be a promising approach to the treatment of stroke patients, and its use should be justified by further experimental and clinical studies.     

Piezoelectricity and prostate cancer: proposed interaction between electromagnetic field and prostatic crystalloids

There is evidence that electromagnetic fields (EMF) play some part in the pathogenesis of prostate cancer, but the pathogenic mechanism remains unknown. The normal prostate gland and both benign and malignant prostate lesions contain abundant calcium/phosphorus crystalloids with various morphologies, which seem to be heterogeneously and diffusely distributed within the gland. We hypothesize that an environmental EMF may result in simultaneous, multidirectional and diffuse compression or expansion of these crystalloids (a piezoelectric effect). This would result in a slight mechanical distortion of the prostate, potentially altering cell behavior and enhancing the expression of specific genes, particularly those involved in suppressing apoptosis. A mathematical model of the cell mechanical effect is presented, and the hypothesis is related to current clinical evidence and to potential validation by critical laboratory tests.     

Toward the development of smart and low cost point-of-care biosensors based on screen printed electrodes

Screen printing technology provides a cheap and easy means to fabricate disposable electrochemical devices in bulk quantities which are used for rapid, low-cost, on-site, real-time and recurrent industrial, pharmaceutical or environmental analyses. Recent developments in micro-fabrication and nano-characterization made it possible to screen print reproducible feature on materials including plastics, ceramics and metals. The processed features forms screen-printed disposable biochip (SPDB) upon the application of suitable bio-chemical recognition receptors following appropriate methods. Adequacy of biological and non-biological materials is the key to successful biochip development. We can further improve recognition ability of SPDBs by adopting new screen printed electrode (SPE) configurations. This review covers screen-printing theory with special emphasis on the technical impacts of SPE architectures, surface treatments, operational stability and signal sensitivity. The application of SPE in different areas has also been summarized. The article aims to highlight the state-of-the-art of SPDB at the laboratory scale to enable us in envisaging the deployment of emerging SPDB technology on the commercial scale.     

Real-time pure shift <Superscript>15</Superscript>N HSQC of proteins: a real improvement in resolution and sensitivity

Spectral resolution in proton NMR spectroscopy is reduced by the splitting of resonances into multiplets due to the effect of homonuclear scalar couplings. Although these effects are often hidden in protein NMR spectroscopy by low digital resolution and routine apodization, behind the scenes homonuclear scalar couplings increase spectral overcrowding. The possibilities for biomolecular NMR offered by new pure shift NMR methods are illustrated here. Both resolution and sensitivity are improved, without any increase in experiment time. In these experiments, free induction decays are collected in short bursts of data acquisition, with durations short on the timescale of J-evolution, interspersed with suitable refocusing elements. The net effect is real-time (t ₂) broadband homodecoupling, suppressing the multiplet structure caused by proton–proton interactions. The key feature of the refocusing elements is that they discriminate between the resonances of active (observed) and passive (coupling partner) spins. This can be achieved either by using band-selective refocusing or by the BIRD element, in both cases accompanied by a nonselective 180° proton pulse. The latter method selects the active spins based on their one-bond heteronuclear J-coupling to ¹⁵N, while the former selects a region of the ¹H spectrum. Several novel pure shift experiments are presented, and the improvements in resolution and sensitivity they provide are evaluated for representative samples: the N-terminal domain of PGK; ubiquitin; and two mutants of the small antifungal protein PAF. These new experiments, delivering improved sensitivity and resolution, have the potential to replace the current standard HSQC experiments.     

Induction of meiotic gynogenesis in ship sturgeon Acipenser nudiventris using UV-irradiated heterologous sperm

Diploid gynogenesis was induced in ship sturgeon Acipenser nudiventris using UV-irradiated sperm from Siberian sturgeon Acipenser baerii. The optimal condition for the retention of the second polar body in ship sturgeon was determined to be 10 min after activation/fertilization in experiments. The temperature of cold shock and its duration were 2.5 °C and 30 min, respectively. A total of 30 gynogens of known parentage from experimental treatments were screened using microsatellite DNA analysis, and uniparental transmission in meiogens was confirmed. The results show that heterologous Siberian sturgeon sperm is applicable as UV-irradiated sperm for the induction of gynogenesis in ship sturgeon. This technique may recover the critically endangered sturgeon species that are becoming extinct.     

Genomic,Proteomic, Morphological,and Phylogenetic Analyses of vB_EcoP_SU10, a Podoviridae Phage with C3 Morphology

A recently isolated phage, vB_EcoP_SU10 (SU10), with the unusual elongated C3 morphotype, can infect a wide range of Escherichia coli strains. We have sequenced the genome of this phage and characterized it further by mass spectrometry based proteomics, transmission electron microscopy (TEM), scanning electron microscopy (SEM), and ultra-thin section electron microscopy. The genome size is 77,327 base pairs and its genes, and genome architecture, show high similarity to the phiEco32 phage genes and genome. The TEM images reveal that SU10 have a quite long tail for being a Podoviridae phage, and that the tail also changes conformation upon infection. The ultra-thin section electron microscopy images of phages at the stage of replication within the host cell show that the phages form a honeycomb-like structure under packaging of genomes and assembly of mature capsids. This implies a tight link between the replication and cutting of the concatemeric genome, genome packaging, and capsid assembly. We have also performed a phylogenetic analysis of the structural genes common between Podoviridae phages of the C1 and C3 morphotypes. The result shows that the structural genes have coevolved, and that they form two distinct groups linked to their morphotypes. The structural genes of C1 and C3 phages appear to have diverged around 280 million years ago applying a molecular clock calibrated according to the presumed split between the Escherichia – Salmonella genera.