Differentiation of the 50B11 dorsal ganglion cells into NGF and GDNF responsive nociceptor subtypes.

The embryonic rat dorsal root ganglion (DRG) neuron-derived 50B11 cell line is a promising sensory neuron model expressing markers characteristic of NGF and GDNF-dependent C-fibre nociceptors. Whether these cells have the capacity to develop into distinct nociceptive subtypes based on NGF- or GDNF-dependence has not been investigated. Here we show that by augmenting forskolin (FSK) and growth factor supplementation with NGF or GDNF, 50B11 cultures can be driven to acquire differential functional responses to common nociceptive agonists capsaicin and ATP respectively. In addition, to previous studies, we also demonstrate that a differentiated neuronal phenotype can be maintained for up to 7 days. Western blot analysis of nociceptive marker proteins further demonstrates that the 50B11 cells partially recapitulate the functional phenotypes of classical NGF-dependent (peptidergic) and GDNF-dependent (non-peptidergic) neuronal subtypes described in DRGs. Further, 50B11 cells differentiated with NGF/FSK, but not GDNF/FSK, show sensitization to acute prostaglandin E2 treatment. Finally, RNA-Seq analysis confirms that differentiation with NGF/FSK or GDNF/FSK produces two 50B11 cell subtypes with distinct transcriptome expression profiles. Gene ontology comparison of the two subtypes of differentiated 50B11 cells to rodent DRG neurons studies shows significant overlap in matching or partially matching categories. This transcriptomic analysis will aid future suitability assessment of the 50B11 cells as a high-throughput nociceptor model for a broad range of experimental applications. In conclusion, this study shows that the 50B11 cell line is capable of partially recapitulating features of two distinct types of embryonic NGF and GDNF-dependent nociceptor-like cells.

Predicting ecological responses of the Florida Everglades to possible future climate scenarios: introduction.

Florida's Everglades stretch from the headwaters of the Kissimmee River near Orlando to Florida Bay. Under natural conditions in this flat landscape, water flowed slowly downstream as broad, shallow sheet flow. The ecosystem is markedly different now, altered by nutrient pollution and construction of canals, levees, and water control structures designed for flood control and water supply. These alterations have resulted in a 50% reduction of the ecosystem's spatial extent and significant changes in ecological function in the remaining portion. One of the world's largest restoration programs is underway to restore some of the historic hydrologic and ecological functions of the Everglades, via a multi-billion dollar Comprehensive Everglades Restoration Plan. This plan, finalized in 2000, did not explicitly consider climate change effects, yet today we realize that sea level rise and future changes in rainfall (RF), temperature, and evapotranspiration (ET) may have system-wide impacts. This series of papers describes results of a workshop where a regional hydrologic model was used to simulate the hydrology expected in 2060 with climate changes including increased temperature, ET, and sea level, and either an increase or decrease in RF. Ecologists with expertise in various areas of the ecosystem evaluated the hydrologic outputs, drew conclusions about potential ecosystem responses, and identified research needs where projections of response had high uncertainty. Resource managers participated in the workshop, and they present lessons learned regarding how the new information might be used to guide Everglades restoration in the context of climate change.

First Report of Laurel Wilt, Caused by Raffaelea lauricola, on Sassafras (Sassafras albidum) in Alabama.

Laurel wilt, caused by Raffaelea lauricola, a fungal symbiont of the redbay ambrosia beetle, Xyleborus glabratus, is responsible for extensive mortality of native redbays (Persea borbonia and P. palustris) in the coastal plains of the southeastern United States (1). The wilt also affects the more widespread sassafras, Sassafras albidum, particularly in areas where diseased redbays are common and populations of X. glabratus are high. Because sassafras stems were thought to lack chemicals that are attractive to the beetle, and sassafras tends to be widely scattered in forests, it was believed that the advance of the laurel wilt epidemic front might slow once it reached the edge of the natural range of redbay, which is restricted to the coastal plains of the Gulf and Atlantic Coasts (2). In July and August of 2011, wilt-like symptoms (i.e., wilted and dead leaves, and streaks of black discoloration in the xylem) were observed on 1 to 10 sassafras trees (15 to 23 cm diameter; 6 to 9 m height) at each of three locations, which were approximately 6 km from one another in Marengo Co., Alabama. Samples of the discolored wood from five trees were plated on malt agar amended with cycloheximide and streptomycin (CSMA), and a fungus morphologically identical to R. lauricola was isolated from each tree (1). For confirmation, a portion of the large subunit (28S) of the rDNA region of three of the isolates was sequenced (3); in each case, the sequence matched exactly that of other isolates of R. lauricola (EU123077) from the United States. Symptomatic trees were found at all three sites when revisited in April 2012, and approximately 20 sassafras trees in various stages of wilt were observed at one location, where only one diseased tree had been noted in 2011. Bolts were cut from the main stem of a symptomatic tree, and eggs, larvae, and adults of X. glabratus were commonly found in tunnels, and R. lauricola was isolated from the discolored xylem. Three container-grown sassafras saplings (mean height 193 cm, mean diameter 2.1 cm at groundline) were inoculated as previously described (1) with conidia (~600,000) from an isolate of R. lauricola. Three additional sassafras saplings were inoculated with sterile, deionized water, and all plants were placed in a growth chamber at 25°C with a 15-h photoperiod. Inoculated plants began to exhibit wilt symptoms within 14 days, and at 30 days all inoculated plants were dead and xylem discoloration was observed. Control plants appeared healthy and did not exhibit xylem discoloration. Pieces of sapwood from 15 cm above the inoculation points were plated on CSMA, and R. lauricola was recovered from all wilted plants but not from control plants. This is the first record of laurel wilt in Alabama and is significant because the disease appears to be spreading on sassafras in an area where redbays have not been recorded (see http://www.floraofalabama.org ). The nearest previously documented case of laurel wilt is on redbay and sassafras in Jackson Co., Mississippi (4), approximately 160 km to the south. The exact source of the introduction of X. glabratus and R. lauricola into Marengo Co. is not known. The vector may have been transported into the area with storms, moved with infested firewood, or shipped with infested timber by companies that supply mills in the area. References: (1) S. Fraedrich et al. Plant Dis. 92:215, 2008. (2) J. Hanula et al. Econ. Ent. 101:1276, 2008. (3) T. Harrington et al. Mycotaxon 111:337, 2010. (4) J. Riggins et al. Plant Dis. 95:1479, 2011.

Susceptibility to Laurel Wilt and Disease Incidence in Two Rare Plant Species, Pondberry and Pondspice.

Laurel wilt, caused by Raffaelea lauricola, has been responsible for extensive losses of redbay (Persea borbonia) in South Carolina and Georgia since 2003. Symptoms of the disease have been noted in other species of the Lauraceae such as the federally endangered pondberry (Lindera melissifolia) and the threatened pondspice (Litsea aestivalis). Pondberry and pondspice seedlings were inoculated with R. lauricola from redbay, and both species proved highly susceptible to laurel wilt. Field assessments found substantial mortality of pondberry and pondspice, but in many cases the losses were not attributable to laurel wilt. R. lauricola was isolated from only 4 of 29 symptomatic pondberry plants at one site, but the fungus was not recovered from three plants at another site. R. lauricola was isolated from one of two symptomatic pondspice plants at one site, and from five of 11 plants at another site, but not from any plant at a third site. Insect bore holes, similar to those produced by Xyleborus glabratus (the vector of laurel wilt), were found in some pondberry and pondspice stems, but adults were not found. Damage caused by Xylosandrus compactus was found in pondberry stems, but this ambrosia beetle does not appear to be a vector of R. lauricola. Xyleborinus saxeseni adults were found in a dying pondspice with laurel wilt, and R. lauricola was recovered from two of three adults. Isolates of R. lauricola from pondberry, pondspice, and X. saxeseni had rDNA sequences that were identical to previously characterized isolates, and inoculation tests confirmed that they were pathogenic to redbay. Because pondberry and pondspice tend to be shrubby plants with small stem diameters, these species may not be frequently attacked by X. glabratus unless in close proximity to larger diameter redbay.

[High-resolution fluorescence microscopy of retinal pigment epithelium using structured illumination]. / Hochauflösende Fluoreszenzmikroskopie des retinalen Pigmentepithels mittels strukturierter Beleuchtung.

BACKGROUND: The accumulation of autofluorescent bodies in retinal pigment epithelium (RPE) cells has an impact on the pathogenesis of retinal diseases, including age-related macular degeneration. While current in vivo fluorescence microscopy allows a lateral resolution of fluorophores in a micrometer range, with ex vivo microscopy a lateral resolution down to 200 nm is possible. For the first time, we used structured illumination microscopy for ex vivo high-resolution fluorescence microscopy of RPE cells. METHODS: Histological sections were prepared from a 68-year-old patient. With epifluorescence microscopy, fluorescent RPE cells were detectable. Structured illumination uses inhomogeneous illumination for resolution of previously nonresolvable structures, similar to the Moiré effect. Images were taken from RPE cells at different excitation wavelengths (488, 568, and 647 nm) and were reconstructed with special software. The different excitation patterns of the fluorescent granules in the RPE cells were colour-coded and analysed. RESULTS: With structured illumination microscopy, autofluorescence signals of RPE cells were detectable, and a lateral resolution of 110 nm could be achieved. Using varying wavelengths, different pigments were excitable. Lipofuscin gave the highest signals, at 488 and 568 nm. The improved resolution showed inhomogeneous intragranular fluorophore patterns. CONCLUSION: Structured illumination microscopy enabled us to generate images of fluorescent structures in RPE cells ex vivo with a lateral resolution of 110 nm. With the use of different excitation wavelengths, intracellular fluorescence patterns in single cell compartments are visible and allow further differentiation.

A comparison of antibiotic resistance in microorganisms isolated from chicken and ostrich faeces in Bulawayo; Zimbabwe

Samples of fresh faeces were obtained from a free-range chicken source; three commercial chicken farms and a commercial ostrich farm; all located around Bulawayo City; Zimbabwe; in order to determine the antibiotic resistance profile of selected bacterial isolates of interest in food-related human infections. Samples were prepared at various dilutions and plated on selective media for Coryneforms; Escherichia coli; Enterococcus faecalis and Pseudomonas. The targeted bacteria were isolated as pure cultures and tested for antibiotic resistance to ampicillin; chloramphenicol; oxytetracycline; sulphonamide; streptomycin and tetracycline. Isolates from the faeces of chickens and ostriches in the commercial farms were found to be generally more resistant to streptomycin; tetracycline and oxytetracycline as compared to those from the free- range chickens. This study emphasizes the need to monitor antibiotic resistance genes in the environment and to curb/curtail antibiotic use for growth promotion in farm animals; particularly in developing countries; as continued use will only add to the growing problem of microbial antibiotic resistance

Leadership. The personal touch.

The government is likely to move from a centralised corporatist form of governance to a more collaborative approach. This will require chief executives to rely on a style of leadership which derives more from personal influence than positional forms of power. It may be time to discuss the future of politically appointed chairs and whether the chief executive should be the sole local leader. The government's commitment to delegating power to regional tiers is likely to have a major impact on the nature of leadership in the NHS.

The effect of growth temperature on Staphylococcus aureus binding to type I collagen.

Many strains of Staphylococcus aureus produce a collagen-binding surface protein that could enable these strains to colonize tissues such as bone. Previous studies indicated that the expression of the collagen receptor varies with growth conditions. We report here that the growth temperature influences the ability of some S. aureus strains to produce this receptor. S. aureus isolates from human, osteomyelitic bone were grown at 37 degrees C and 42 degrees C and tested for agglutination of collagen-coated latex beads. Binding by 42 degrees C grown cells was significantly reduced in five of the seven isolates studied, including a complete loss of collagen binding in three of these isolates. In an 125I-collagen-binding assay, the binding ability of one of these isolates, strain #16, was 20-fold lower if grown at 42 degrees C. Reduced collagen binding by this isolate could be demonstrated after only two cell divisions at 42 degrees C and the cells regained the ability to bind collagen when shifted back to 37 degrees C. Sodium dodecyl sulfate (SDS)-PAGE confirmed the presence of proteins at 117 kDa in strain #16 and 135 kDa in SMH which were absent following growth at 42 degrees C. Chicken IgG, specific for the 117 kDa protein, was found to react in immunoblot assays with these proteins as well as a protein of 135 kDa extracted from S. aureus Cowan 1. The antibody did not react with proteins extracted from non-binding strains. Strains #15 and #21, collagen-binders at both 37 degrees C and 42 degrees C, produced immunoreactive proteins at 110 and 135 kDa, respectively, in lysates from cells grown at both temperatures. Antibody against a recombinant form of a previously characterized collagen receptor was used to confirm cross-reactivity with these novel collagen receptors. These data suggest that the ability to produce the collagen receptor is temperature sensitive in some S. aureus strains associated with osteomyelitis. It is proposed that a better understanding of the environmental effects on collagen receptor production could enhance our understanding of staphylococcal infections in bone and joints.

Managing the new NHS: breathing new life into the NHS reforms.

The essence of the NHS reforms is that they bring market forces to bear on organisations providing public services, while allowing those organisations more freedom to respond in ways that will improve the efficiency, effectiveness, and appropriateness of their services. The new structural changes to the NHS--a leaner management executive and fewer, slimmer regions--could be used either to strengthen these features of the reforms or frustrate them by allowing ministers and top management to intervene even more at local level and "overmanage" the market. To ensure that the aims of the reforms are not frustrated ministers and the management executive must restrict themselves to laying down clear strategies and then allow purchasers and providers to meet those strategies in their own ways. They also need to ensure that the whole NHS can learn and benefit from local experimentation and devise ways of managing the crises that will inevitably arise; otherwise they might be tempted to become involved in managing the market at too local a level, and the NHS will suffer the worst of both worlds: stifling bureaucracy at the top and parochial self interest locally.

Binding of a Staphylococcus aureus bone pathogen to type I collagen.

We contrasted the collagen-binding potential of the experimental osteomyelitis pathogen, Staphylococcus aureus strain SMH, to several other strains. These included Cowan 1 (binder), Wood 46 (non-binder) and six capsular variants. These measurements were made using an 125I-collagen binding assay. Formalin-killed S. aureus SMH strongly bound commercial type I iodinated collagen (dissociation constant, Kd = 2 x 10(-9) M). The extent of binding was similar to Cowan 1. Binding was saturable and not inhibited by 100 mM solutions of D-glucose, D-galactose, D-mannose, methyl-alpha-L-fucopyranoside, L-hydroxyproline or L-glycine. D-lactose gave moderate inhibition of binding to collagen, and L-fucose was strongly inhibitory. Trypsinized SMH did not bind collagen. None of four Ruthenium-red-staining staphylococci (encapsulated) avidly bound type I collagen. The encapsulated Smith strain, for example, did not bind to collagen but its capsule-negative variant, Smith compact, showed extensive binding. Three of five non-encapsulated S. aureus were strong collagen binders. These data suggest that the prototype bone pathogen binds to the major protein component of bone's extracellular matrix. Collagen-binding is promoted by protein adhesin(s), not capsule. The latter, in fact, appeared to interfere with this interaction. Binding was inhibited by solutions containing the simple monosaccharide, L-fucose.

Endotoxin enhancement of toxic shock syndrome toxin 1-induced secretion of interleukin 1 by murine macrophages.

The purpose of this study was to determine whether endotoxin could augment toxic shock syndrome toxin 1 (TSST-1)-induced production of interleukin 1 (IL-1) by murine macrophages. Macrophages from C3H/HeJ or C57Bl/6 mice were stimulated with purified TSST-1 alone or in combination with lipopolysaccharide (LPS). A dramatic synergistic thymocyte-proliferative response was induced by supernatants from C57Bl/6 macrophages stimulated with both TSST-1 and LPS. No enhanced response was induced by supernatants from C3H/HeJ macrophages. A portion of the enhanced response induced by C57Bl/6 macrophage supernatants was attributed to synergism between IL-1 and residual TSST-1 in the thymocyte assay. The addition of monoclonal antibody to TSST-1 to the supernatants eliminated the effects of residual TSST-1 in the thymocyte assay and demonstrated a synergistic induction of IL-1. These data (1) show that LPS can enhance macrophage responsiveness to TSST-1; (2) suggest that TSST-1 not only induces IL-1 secretion but also enhances target cell responsiveness to IL-1; and (3) further support the role of IL-1 in the pathogenesis of toxic shock syndrome.

Passive protection of rabbits infected with toxic shock syndrome-associated strains of Staphylococcus aureus by monoclonal antibody to toxic shock syndrome toxin 1.

The effectiveness of passive immunization was assessed in an infection model of toxic shock syndrome (TSS) in which monoclonal antibody to TSS toxin 1 (TSST-1) was administered intravenously to rabbits. Previously implanted infection chambers were inoculated with Staphylococcus aureus strains RN4710 and D4508. The former strain carries the TSST-1 gene on plasmid pRN6201; the latter is a TSST-1-negative clinical isolate obtained from a patient with nonmenstrual TSS. Purified monoclonal antibody, MAb 8-5-7 (IgG), was administered in two doses of approximately 1.25 mg each 24 hours before and 24 hours after infection. MAb 8-5-7 provided complete protection against both the TSS-like syndrome and the mortality that occurred in unprotected rabbits infected with strain RN4710 but did not provide complete protection in rabbits infected with strain D4508; three of the five rabbits either displayed signs of illness or died despite treatment. Western-blot analyses of the extracellular proteins produced by strains RN4710 and D4508 that used MAb 8-5-7 as a probe revealed that only TSST-1 produced by RN4710 reacted with the antibody. Thus, if MAb 8-5-7 partially protected animals against infections with strain D4508, the protection appears to have been nonspecific.

Protection of rabbits in an infection model of toxic shock syndrome (TSS) by a TSS toxin-1-specific monoclonal antibody.

An anti-TSST-1-specific monoclonal antibody (MAb 8-5-7) was tested for its protective capacity in a rabbit infection model to toxic shock syndrome (TSS). The challenge strain of Staphylococcus aureus (RN4710), which contained a plasmid encoding TSS toxin-1, was introduced into previously implanted chambers. Purified monoclonal antibody (1.25 mg of immunoglobulin G) administered parenterally 1 day before and 1 day after initiation of infection provided complete protection against the TSS-like syndrome and the mortality which occurred in unprotected rabbits.

Synergistic induction of interleukin-1 by endotoxin and toxic shock syndrome toxin-1 using rat macrophages.

We studied interleukin-1 (IL-1) secretion by rat peritoneal exudate macrophages stimulated with purified toxic shock syndrome toxin-1 (TSST-1). TSST-1 was observed to be a more potent inducer of IL-1 than was endotoxin. The induction of IL-1 secretion by TSST-1 was not blocked by polymyxin B but could be blocked by monoclonal antibodies directed against TSST-1. Synergistic induction of IL-1 was observed when the cells were stimulated with TSST-1 and endotoxin. The sequence of addition was found to be important for the synergistic response. Enhanced IL-1 production was observed only when macrophages were exposed to endotoxin before or simultaneously with TSST-1. Prior exposure of macrophages to TSST-1 had no enhancing effect on endotoxin-induced IL-1 secretion. We conclude that stimulation of the macrophage by endotoxin enhances the responsiveness of the cells to TSST-1 and may thereby play a role in the pathogenesis of toxic shock syndrome.