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Original Research ARTICLE
Deferiprone and Gallium-Protoporphyrin Have the Capacity to Potentiate the Activity of Antibiotics in Staphylococcus aureus Small Colony Variants
- 1Department of Surgery, Otolaryngology Head and Neck Surgery, Basil Hetzel Institute for Translational Health Research, The Queen Elizabeth Hospital, University of Adelaide, Adelaide, SA, Australia
- 2School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, SA, Australia
- 3Adelaide Biofilm Test Facility, Sansom Institute for Health Research, University of South Australia, Adelaide, SA, Australia
- 4Department of Otolaryngology Head and Neck Surgery, Tianjin First Center Hospital, Tianjin, China
- 5ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Adelaide, SA, Australia
- 6Laboratory of Pharmaceutical Microbiology, Ghent University, Ghent, Belgium
Small colony variants (SCVs) of bacteria like Staphylococcus aureus are characterized by a reduced colony size and are linked to increased antibiotic tolerance and resistance. Their altered expression of virulence factors, slow growing properties and their ability to form biofilms make the eradication of SCVs challenging. In the context of biofilm-related infectious diseases involving S. aureus SCVs, a therapy targeting bacterial iron metabolism was evaluated. The combination of the iron-chelator deferiprone (Def) and the heme-analog gallium-protoporphyrin (GaPP), in solution and incorporated in a surgical wound gel, was tested for activity against planktonic and sessile SCVs. To this end, the activity of Def-GaPP was assessed against planktonic S. aureus SCVs, as well as against in vitro and in vivo biofilms in the colony biofilm model, an artificial wound model and a Caenorhabditis elegans infection model. While Def alone failed to show substantial antibacterial activity, GaPP and the combination of Def-GaPP demonstrated concentration- and strain-dependent antibacterial properties. Specifically, the Def-GaPP combination significantly reduced the bacterial load in an artificial wound model and increased the survival of S. aureus SCV infected C. elegans. When Def-GaPP were combined with gentamicin or ciprofloxacin, the triple combinations exceeded the antibiofilm activity of the individual compounds in the colony biofilm model. In targeting bacterial iron metabolism, Def-GaPP showed significant activity against planktonic and sessile SCVs. Moreover, Def-GaPP could potentiate the activity of gentamicin and ciprofloxacin. Delivered in a wound healing gel, Def-GaPP showed promise as a new topical strategy against infections with S. aureus SCVs.
Small colony variants (SCVs) are naturally occurring bacteria derived from a parent strain characterized by a small colony morphology (~10% the size of the parent strain colony), slow growth rate, altered virulence factors and increased antibiotic tolerance or resistance (Proctor et al., 2006). The switch to a phenotypic altered strain can be inheritable or transient (Johns et al., 2015). Staphylococcus aureus SCVs are frequently non-pigmented, non-hemolytic, and dependent on external growth factors like menadione, hemin, and thymidine; they are able to survive inside eukaryotic cells, including human macrophages (von Eiff et al., 1997b; Proctor et al., 2006; Garcia L. et al., 2012). Due to their intracellular lifestyle, S. aureus SCVs can escape the immune attack and are protected against antibiotics leading to persistence of disease. SCVs are associated with antibiotic-refractory and recalcitrant infections, such as chronic rhinosinusitis, respiratory tract infections in cystic fibrosis, osteomyelitis, chronic wounds, or implant infections (Proctor et al., 2006; Garcia et al., 2013; Tan et al., 2014). The recovery of SCVs in routine clinical investigations requires special nutrients and prolonged culture, making SCV isolation and identification difficult (von Eiff, 2008). Prolonged treatment regimens with a variety of antibiotics are required to treat SCV-associated infections, often combined with surgical interventions. However, clinical outcomes are frequently unsatisfying due to treatment failure and recurrence of disease (Garcia et al., 2013).
Worsening the situation, SCVs can be induced by medical therapies e.g., by exposure to antibiotics such as gentamicin, or disinfectants such as triclosan (von Eiff et al., 1997a; Bayston et al., 2007). It is furthermore known that sub-therapeutic antibiotic exposure can trigger biofilm formation of S. aureus SCVs and their parent strains, further complicating treatment (Costerton et al., 1995; Mitchell et al., 2010a,b). Despite the clinical significance there is little knowledge concerning S. aureus SCV biofilms and their susceptibility to antibiotics. Innovative treatment approaches, based on compounds with a different mode of action, such as disrupting bacterial iron metabolism (Richter et al., 2017a), may be a strategy worth approaching. S. aureus SCVs, like all bacteria, rely on iron for growth and survival (Andrews et al., 2003), hence, the iron metabolism could be an interesting therapeutic target.
In the present study, the antimicrobial activity of a treatment combining the iron-chelator deferiprone (Def) and the heme analog gallium-protoporphyrin (GaPP) was assessed against planktonic and biofilm-associated SCVs.
Materials and Methods
Bacterial Strains
Bacterial strains were collected from the sinonasal cavities of chronic rhinosinusitis patients. Ethics approval was obtained from The Queen Elizabeth Hospital Human Research Ethics Committee, Woodville, SA, Australia. Strains included one S. aureus clinical isolate (parent strain P1), which was prolonged subcultured at MIC or higher concentrations of gentamicin (≥2 μg/ml). This induced small colony variants (SCV1) that featured elevated gentamicin tolerance. Another strain was a clinically isolated S. aureus small colony variant (SCV2).
Characteristics of Bacterial Strains
Catalase, Coagulase, and Hemolytic Activity
Catalase and coagulase activity of bacterial strains were determined by suspending cells in saline in a glass tube. The catalase activity was observed by gas formation following addition of hydrogen peroxide. The coagulase activity was determined by clumping of bacterial cells after addition of plasma. Hemolytic activity was determined by streaking out bacteria on sheep blood agar (Oxoid, Thermo Fisher Scientific, Scoresby, VIC, Australia). Following incubation at 37°C for 24 h the presence of a hemolysis zone was observed.
Auxotrophy Determination
The auxotrophy type of bacteria was determined as previously described (van de Rijn and Kessler, 1980). Briefly, a bacterial suspension adjusted to 1.0 McFarland units (~3 × 108 CFU/ml) was diluted 1:100 in physiological saline. One hundred microliters were spread on chemically defined medium agar (van de Rijn and Kessler, 1980). Sterile disks were infiltrated with 10 μl of hemin, menadione, and thymidine, respectively, and placed on top of the agar. Plates were incubated for 48 h at 37°C. According to the growth zones around the disks auxotrophy was determined.
MIC Determination
The colony suspension and broth microdilution method (Wiegand et al., 2008) were used to determine the MIC of deferiprone (Def, 3-hydroxy-1,2-dimethylpyridin-4(1H)-one, Sigma, Castle Hill, NSW, Australia) and gallium-protoporphyrin IX (GaPP, Frontier Scientific, Logan, UT) over 48 h. The concentrations ranged from 0.08 to 40 mM Def (i.e., 10.8–5,568 μg/ml) and 0.1–50 μg/ml GaPP. In addition, the MICs of ciprofloxacin (Cip), gentamicin (Gent), mupirocin (Mup), doxycycline (Doxy), chloramphenicol (Chlor), cephalexin (Ceph), vancomycin (Van), amoxicillin (Amoxi), and streptomycin (Strep) were determined (concentration range 0.06–32 μg/ml). All compounds were purchased from Sigma unless stated differently.
Bacterial Growth
Bacteria were suspended in tryptone soya broth (Oxoid) and adjusted to an OD 600 of 0.01. Bacterial growth was measured in a 96-well plate over 40 h at 37°C using an EnSpire Multimode Plate Reader (Perkin Elmer, Waltham, MA). Loops were taken after 24 and 40 h to determine the colony morphology.
Hydrogel Preparation
Hydrogels were prepared by adequate mixing of dextran-aldehyde, succinyl-chitosan, and a buffer solution, as previously described (Richter et al., 2017b). Def (20 mM) and/or GaPP (100 or 500 μg/ml) were incorporated in the gel and compared to blank gel and antibiotic loaded gel including 5 μg/ml ciprofloxacin (Cip) and 100 μg/ml gentamicin (Gent). Cip and Gent were chosen due to their clinical relevance as antibiotic therapies for respiratory tract, skin, blood, bone, and soft tissue infections. Furthermore, fluoroquinolone antibiotics (such as Cip) were described as being highly effective against SCVs in vitro and in vivo (Garcia L. et al., 2012; Garcia et al., 2013).
In vitro Activity in the Colony Biofilm Model
A bacterial suspension was prepared in 0.9% saline and adjusted to 1.0 McFarland units (~3 × 108 CFU/ml). After diluting bacteria 1:1,000 in tryptone soya broth, 2 μl were spotted on UV-sterilized Whatman polycarbonate membrane filters (0.2 μm pore size, GE Healthcare, Little Chalfont, UK) and placed on tryptone soya agar (Merritt et al., 2005). Following biofilm formation after 24 h incubation at 37°C, the membrane filters were transferred onto AB trace agar (minimal growth agar including 0.5% glucose and 0.5% peptone). One hundred microliters of freshly prepared hydrogel was placed on biofilms and incubated for 2.5 days at 37°C. The filter membranes were then aseptically transferred to new AB trace agar plates and incubated for 2.5 days at 37°C. Finally, the filters were collected in PBS to extract bacteria by vortexing (1 min) and sonication (15 min), prior to serial dilutions and plating on tryptone soya agar for CFU counting and colony morphology determination after 3 days incubation at 37°C.
The antibiofilm effect of drug loaded gels was rated using the Bliss Independence Model (Morones-Ramirez et al., 2013; Thangamani et al., 2015). The synergy of treatment combinations (Def-GaPP100, Def-GaPP100-Cip, Def-GaPP100-Gent, Def-GaPP500) was calculated according to the following equation, with values above zero corresponding to synergistic effects.
S = synergistic effect, a = Log10 of biofilm after exposure to compound a, b = Log10 of biofilm after exposure to compound b, ab = Log10 of biofilm after exposure to treatment combination ab, MG = Log10 of untreated biofilm (maximum growth).
In vitro Activity in an Artificial Wound Model
Hyaluronic acid (1.20–1.80 MDa, Lifecore Biomedical, Chaska, MN) and collagen (Corning Incorporated, Corning, NY) were used to prepare an artificial dermis (Brackman et al., 2016), which was immersed in lyophilized bovine plasma, 19 ml Bolton broth (Oxoid), 1 ml horse blood and 10 IU of heparin. Subsequently, 10 μl of an overnight culture adjusted to 1 × 106 CFU/ml was spotted on top of the dermis and incubated for 24 h at 37°C. The formed biofilms were exposed to 150 μl gel (blank gel, Def, GaPP 500, Def-GaPP500, and Cip gels) for 24 h at 37°C. Following a washing step, the dermis was immersed in 10 ml of 0.9% saline to recover the bacteria by vortexing and sonication (alternating cycles of 3 × 30 s), prior to serial dilutions and plating for CFU counting. In addition, the colony morphology was determined.
In vivo Activity in a C. elegans Infection Model
Synchronized nematodes, Caenorhabditis elegans AU37 (glp-4; sek-1), were grown to L4 stage, suspended in OGM medium (95% M9 buffer, 5% brain heart infusion broth, 10 μg/ml cholesterol) and added into 96-well plates with at least 20 worms per well (Brackman et al., 2011). Nematodes were infected with 25 μl of an overnight culture adjusted to 2 × 109 CFU/ml in OGM medium and exposed to 25 μl of treatment (Def 20 mM, GaPP 500 μg/ml or a combination of both). Uninfected nematodes in OGM medium as well as infected but untreated nematodes were used as controls. The number of viable and dead nematodes was assessed every 24 h over 3 days incubation at 25°C. Subsequently, nematodes were first washed in M9 buffer containing 1 mM sodium azide, then washed in PBS prior to counting. The nematodes were mechanically disrupted by vortexing the worms in microtubes with 1.0 mm silicon carbide beads for 10 min (BioSpec Products, Bartlesville, OK). Serial dilutions of the supernatants were plated (tryptone soya agar with 7.5% NaCl) for CFU counting and colony morphology determination.
Statistics and Software
All experiments were conducted in triplicate and are presented as mean ± standard deviation (SD) or mean ± standard error of the mean (SEM). Results were analyzed using two-way analysis of variance with Dunnett's test (GraphPad Prism version 7.02, GraphPad Software, La Jolla, CAL). Statistical significance was assessed at the 95% confidence level.
Characteristics of Bacterial Strains
The colony morphology of bacterial strains is shown in Figure 1 and the growth curves are displayed in Figure 2. A summary of bacterial characteristics is shown in Table 1.
Figure 2. Growth curves of small colony variant SCV1 (green triangles), SCV2 (red dots) and parent strain P1 (blue diamonds).
Table 1. Characteristics of small colony variant SCV1, SCV2, and parent strain P1, including catalase, coagulase and hemolytic activity, auxotrophy type, as well as MICs (in μg/ml) of deferiprone (Def), gallium-protoporphyrin (GaPP), the combination of both compounds, ciprofloxacin (Cip), gentamicin (Gent), mupirocin (Mup), doxycycline (Doxy), chloramphenicol (Chlor), cephalexin (Ceph), vancomycin (Van), amoxicillin (Amoxi), and streptomycin (Strep).
SCV1 was observed to be a catalase positive, coagulase positive, and hemolysis positive strain with hemin/menadione auxotrophy. SCV2 was determined as a catalase negative, coagulase positive, and hemolysis negative strain with thymidine auxotrophy. P1 was identified to be catalase positive, coagulase negative, and hemolysis positive with hemin/menadione auxotrophy. As depicted in Figure 2, during 24 h SCV1 and SCV2 showed a slower growth rate and a lower OD 600 value than the parent strain P1. While SCV1 reached stationary and decline phase after 24 h, SCV2 continued to grow reaching an OD 600 of 0.95 after 40 h. Loops taken after 24 h revealed a small colony morphology for both SCV1 and SCV2, while after 40 h SCV2 presented as a mix of small colonies and very few normal sized colonies. SCV1 showed a small morphology after 40 h.
SCV1 and SCV2 were 4- and 16-fold more susceptible to Def and 4- and 8-fold less susceptible to Cip and Gent compared to the parent strain P1. SCV1 and P1 had low MICs for GaPP (6.25 and 12.5 μg/ml, respectively), while SCV2 was observed to have a MIC above 50 μg/ml for GaPP. The MICs for the Def-GaPP combination were typically lower than the MICs for individual compounds, however, the extent of this difference was strain-dependent. As displayed in Table 1, SCV1 was susceptible to Mup and Van, and showed increasing MIC values for Ceph, Doxy, and Chlor, and was not susceptible to Amoxi and Strep. SCV2 was susceptible to Doxy, less susceptible to Chlor and not susceptible to Mup, Ceph, Van, Amoxi, and Strep. P1 showed low MIC values for Doxy and Mup, and increasing MICs for Van, Ceph, Amoxi, and Chlor, and was not susceptible to Strep.
Colony Biofilm Model
The blank gel and the Def gel showed no antibiofilm activity (data not shown), while the GaPP gel demonstrated a concentration- and strain-dependent effect. Gel loaded with a low concentration of GaPP (100 μg/ml) showed a log10 reduction of 1.7 and 1.8 against biofilms of SCV1 and its parent strain P1, respectively, but no antibiofilm activity against SCV2 (data not shown), while at 500 μg/ml GaPP showed a log10 reduction of 4.3, 1.4, and 2.0 in SCV1, SCV2, and P1 biofilms (Figure 3). Cip and Gent loaded gels were observed to have only a minor effect against SCV1, SCV2, and P1 (log10 reduction of 0.4, 0.1, and 1.1, respectively for Cip and 0.2, 0.8, and 1.1, respectively for Gent). A small colony morphology of both SCV1 and SCV2 has been observed when analyzing CFUs after treatment exposure.
Figure 3. Log10 reduction of small colony variant SCV1 (A), SCV2 (B) and parent strain P1 (C) colony biofilms after exposure to drug loaded hydrogels compared to untreated control. 1: Gentamicin (Gent) 100 μg/ml (light gray), 2: Ciprofloxacin (Cip) 5 μg/ml (purple), 3: Deferiprone (Def, 20 mM)-Gallium-protoporphyrin (GaPP) 100 μg/ml (blue), 4: Def-GaPP100-Cip (black), 5: Def-GaPP500 (red), 6: GaPP 500 (orange), 7: Def-GaPP100-Gent (dark gray). Data represent the mean ± SD of three biological replicates. Op < 0.001; #p < 0.0001; ns-not statistically significant.
The hemin auxotroph SCV1 (Figure 3A) showed increased susceptibility to gel loaded with Def-GaPP100-Cip, Def-GaPP100-Gent, GaPP 500, and Def-GaPP500 (log10 reduction of 3.4, 5.4, 4.3, and 4.4, respectively), and decreased susceptibility to monotherapy with Cip or Gent compared to its parent strain P1. Interestingly, Def-GaPP100 combined with Gent showed a high degree of synergy (Figure 4) and significant activity against the highly Gent-tolerant SCV1 compared to Gent alone and Def-GaPP100 (log10 reduction of 5.4 for Def-GaPP100-Gent vs. 0.2 for Gent and 1.3 for Def-GaPP100, p < 0.0001).
Figure 4. Synergy of treatment combinations against small colony variant SCV1, SCV2 and parent strain P1 colony biofilms. Deferiprone 20 mM (Def)-Gallium-protoporphyrin (GaPP) 100 μg/ml (blue circles), Def-GaPP500 μg/ml (red squares), Def-GaPP100-ciprofloxacin 5 μg/ml (black triangles), Def-GaPP100-gentamicin 100 μg/ml (gray diamonds). The higher the value the higher the degree of synergy.
The thymidine auxotroph SCV2 (Figure 3B) showed elevated susceptibility to gel incorporating Def-GaPP100-Cip, Def-GaPP100-Gent, GaPP 500, and Def-GaPP500 (log10 reduction of 3.1, 1.7, 1.4, and 2.5, respectively), and no susceptibility to Cip or Def-GaPP100. Notably, Def-GaPP100 combined with Cip showed high synergy (Figure 4) and significant activity against SCV2, manifestly exceeding the effect of the individual compounds and Def-GaPP100 (log10 reduction of 3.1 for Def-GaPP100-Cip vs. 0.1 for Cip and 0.1 for Def-GaPP100, p < 0.0001).
Against the hemin auxotroph P1 (Figure 3C) the combination of Def-GaPP100 gel with Cip or Gent showed significantly higher activity than the antibiotics alone (log10 reduction of 1.8 for Def-GaPP100-Cip vs. 1.1 for Cip, p < 0.001; and 1.9 for Def-GaPP100-Gent vs. 1.1 for Gent, p < 0.0001). However, no synergistic effect was observed compared to the Def-GaPP100 gel (1.8 log10 reduction). The highest activity against P1 was achieved with Def-GaPP500 gel (2.4 log10 reduction).
Macroscopic Biofilm Analysis
The antibiofilm activity of loaded hydrogels was macroscopically analyzed over 5 days of treatment exposure. All biofilms grew extensively when exposed to blank gel and gel incorporating Def (not shown), Cip and Gent (Figure 5). A concentration- and strain-dependent antibiofilm effect was apparent after exposure to gels incorporating GaPP (not shown), Def-GaPP, Def-GaPP-Cip, and Def-GaPP-Gent. While Def-GaPP100 gel moderately inhibited bacterial growth, the combination of Def-GaPP100 with either Cip or Gent resulted in a substantial antibiofilm effect (Figure 5).
Figure 5. Inhibitory effect of drug loaded hydrogels on biofilms after 5 days exposure. Elevated biofilm inhibition was observed for gels containing deferiprone, gallium-protoporphyrin and ciprofloxacin or gentamicin (DGCip, DGGent). Strains used: Small colony variant SCV1, SCV2 and parent strain P1. Hydrogels- B, Blank control gel; Cip, Ciprofloxacin 5 μg/ml; Gent, Gentamicin 100 μg/ml; DG, Deferiprone 20 mM-Gallium-protoporphyrin 100 μg/ml; DGGent, Def-GaPP100-Gent; DGCip, Def-GaPP100-Cip.
Artificial Wound Model
Biofilms grown on an artificial dermis were exposed to drug loaded hydrogels to determine the antibiofilm activity in an in vitro wound model (Figure 6). The visual analysis of CFUs after treatment exposure confirmed a small colony morphology for the majority of both SCV1 and SCV2. The untreated control and the blank gel showed similar growth of all biofilms, indicating no antibiofilm effect of the blank gel. The Def gel demonstrated substantial antibiofilm activity against SCV1 (log10 reduction of 0.9), but failed to be effective against P1 and SCV2. The GaPP 500 gel showed minor antibiofilm activity with a 0.2–0.4 log10 reduction. In contrast, Def-GaPP500 gel showed significant antibiofilm effects against SCV1, SCV2, and P1 with a 1.4, 1.0, and 0.9 log10 reduction, respectively, thereby demonstrating significantly higher activity than the individual compounds (p < 0.05–0.0001) and slightly higher activity than Cip gel (Figure 6).
Figure 6. Effects of hydrogels in an artificial wound model compared to untreated control. Log10 reduction of small colony variant SCV1, SCV2, and parent strain P1 after exposure to hydrogels loaded with ciprofloxacin 5 μg/ml (purple), deferiprone 20 mM (Def, gray), gallium-protoporphyrin 500 μg/ml (GaPP, orange), and Def-GaPP500 (red). Data represent the mean ± SD of three biological replicates. *p < 0.05; **p < 0.01; #p < 0.0001.
In vivo Infection Model in C. elegans
Nematodes were infected with bacteria and their survival rate was determined with and without Def, GaPP 500, or Def-GaPP500 treatment (Figure 7). The worm killing was strain-dependent with 75, 25, and 57% survival in SCV1, SCV2, and P1 infected worms, respectively, while uninfected controls showed 88% survival over 3 days. When worms were exposed to Def, 45, 73, and 71% of SCV1, SCV2, and P1-infected nematodes survived, while 73, 48, and 81% infected worms survived when treated with GaPP 500. The combination of Def-GaPP500 showed a similar survival rate in SCV1 and P1 infected worms as GaPP 500 alone (71 and 87% survival, respectively). In contrast, the survival rate of SCV2 infected worms was substantially higher (86% survival) when treated with Def-GaPP500 compared to the individual treatments. Compared to the uninfected control the Def-GaPP500 treatment achieved similar survival rates in SCV2 and P1 infected worms. Furthermore, the survival of SCV1 and P1 infected worms after GaPP 500 treatment was not different to uninfected controls (Figure 7).
Figure 7. C. elegans survival (%) over 3 days in uninfected controls (light gray) and after infection (black bars) with small colony variant SCV1 (A), SCV2 (B) or parent strain P1 (C) and treatment with loaded hydrogels: deferiprone 20 mM (Def, dark gray), gallium-protoporphyrin 500 μg/ml (GaPP, orange), and Def-GaPP500 (red). Data represent the mean ± SEM of at least six biological replicates. **p < 0.01; #p < 0.0001.
Following 3 days of infection the bacterial load per worm was quantified (Figure 8) by enumeration of CFU. Small colony phenotypes of SCV1 and SCV2 were observed. Consistent with results in the colony biofilm model and wound model, the treatment with Def alone showed no significant effect and failed to reduce the CFU per worm. In contrast, both GaPP 500 and Def-GaPP500 showed a significant (p < 0.05) reduction of the bacterial load, resulting in a log10 of 2.0 for GaPP and 2.3 for Def-GaPP in SCV1 infected worms (SCV1 infection control: log10 of 3.6 CFU/worm), 2.9 and 2.5 in P1 infected worms (P1 infection control: log10 of 3.6 CFU/worm), and 3.0 and 2.8 in SCV2 infected worms (SCV2 infection control: log10 of 4.5 CFU/worm).
Figure 8. Log10 of CFU per C. elegans worm after 3 days infection (black bars) with small colony variant SCV1 (A), SCV2 (B) or parent strain P1 (C) and treatment with drug loaded hydrogels- Def: deferiprone 20 mM (gray), GaPP, gallium-protoporphyrin 500 μg/ml (orange) and DG, Def-GaPP500 (red). Data represent the mean ± SD of at least 6 biological replicates. *p < 0.05.
S. aureus SCVs in planktonic and biofilm form have a global significance in the clinical environment being associated with treatment failure and recurrence of disease (Proctor et al., 2006; Garcia et al., 2013). Treatments are mostly based on antibiotics, however, the low growth rate, reduced antibiotic susceptibility and emerging resistance of SCVs pose a challenge for efficient medical therapies. In the present study, an alternative treatment that relies on the disruption of bacterial iron metabolism was evaluated. As previously described, the iron chelator Def and the heme analog GaPP show synergistic effects against S. aureus biofilms in vitro (Richter et al., 2016). Herein, the activity of Def-GaPP against planktonic and biofilm-associated SCVs and a parent strain with elevated antibiotic tolerance/resistance was evaluated in vitro and in vivo.
The Def-GaPP treatment exhibits antimicrobial activity based on the initial iron-chelation by Def, followed by iron depletion-induced upregulation of the bacteria's iron acquisition systems (Haley and Skaar, 2012). The latter are exploited by the heme analog GaPP mimicking heme as the preferred iron source of S. aureus (Stojiljkovic et al., 1999; Skaar et al., 2004; Weinberg, 2009). Inside bacteria, GaPP disrupts the iron metabolism vital for bacterial growth, survival, and virulence (Weinberg, 2009; Skaar, 2010). Unlike heme, GaPP lacks the ability to transfer electrons, hence, GaPP cannot be part of redox reactions required for respiration, ATP production and DNA synthesis. Furthermore, GaPP cannot be cleaved by bacterial enzymes and cannot be utilized as nutrient source leading to starvation. In addition, GaPP is able to block efflux pumps essential for heme homeostasis resulting in an intracellular accumulation of redox-active molecules (Stojiljkovic et al., 2001; Reniere et al., 2007). The subsequent antibacterial effects are based on starvation, limited respiration and elevated production of reactive oxygen species contributing to DNA and protein damage and ultimately cell death.
As shown in the present study, the combination of Def and GaPP exhibited significant antibacterial and antibiofilm activity against clinical isolates of S. aureus SCVs and a parent strain. In the colony biofilm model, where compounds were delivered in a surgical wound gel, a dose-dependent antibiofilm effect of GaPP and Def-GaPP was observed (Figures 3, 5). A colony biofilm comprises of a heterogeneous consortium of sessile cells with a mixture of metabolic active bacteria on the biofilm surface and metabolic retarded/inactive bacteria on the inside. The metabolic activity arises due to different oxygen levels (aerobic conditions on the biofilm surface, micro-aerobic/anaerobic inside the biofilm) and different nutrient availability according to the location bacteria occupy. While sessile bacteria on the biofilm surface can relatively easily be targeted, bacteria deeper in the biofilm are less exposed to compounds and show reduced susceptibility (Merritt et al., 2005). The blank gel and Def gel are thought to interfere only with the biofilm surface by attaching to bacterial cell wall proteins and depriving nutrients, respectively, resulting in minor antibiofilm activity. Likewise, gels incorporating low concentrations (100 μg/ml) of GaPP showed limited activity, indicating an insufficient GaPP penetration into the colony biofilm and/or a suboptimal GaPP concentration. A higher GaPP concentration in gels (GaPP 500, Def-GaPP500) resulted in higher antibiofilm activity. It was observed that the presence of Def was crucial to facilitate a significant activity of GaPP against SCV2 biofilms, which is likely based on a Def-induced upregulation of heme acquisition systems elevating GaPP uptake into bacteria for an improved antibiofilm effect. Another reason for differences in susceptibility of the strains used in this study can rely on auxotrophy (Garcia et al., 2013). SCVs can be classified in two types of auxotrophs, namely hemin/menadione auxotrophs and thymidine auxotrophs. This in return affects the susceptibility toward antibiotics and other antibacterial compounds (Garcia et al., 2013). SCV2 was observed to be auxotrophic for thymidine, hence, less dependent on hemin/iron which can explain the poor GaPP susceptibility. SCV1 was shown to be hemin/menadione auxotrophic, thus, more susceptible to the iron depriving and heme mimicking Def-GaPP treatment.
SCVs are electron-transport-defective strains with a reduced transmembrane potential, which impedes the penetration and activity of membrane active compounds and some antibiotic classes, such as aminoglycosides and antifolate agents (von Eiff et al., 1997a; McNamara and Proctor, 2000; Baumert et al., 2002). Therefore, Gent was expected to show low activity against both planktonic SCVs as confirmed in Table 1, and sessile SCVs as seen in Figures 3, 5. Surprisingly, when Gent was combined with Def-GaPP, the triple combination showed significant antibiofilm activity (Figures 3, 5), indicating a potentiation of Gent by Def-GaPP even against the Gent-tolerant SCV1 strain. This result implies that by disrupting bacterial iron metabolism Def-GaPP made the bacteria vulnerable and increased their susceptibility to Gent for a synergistic antibiofilm effect as confirmed in Figure 4. Similar results were observed for the treatment with Cip. In line with the literature, planktonic SCVs showed higher MIC values for Cip than the parent strain (Idelevich et al., 2011) and biofilm-associated SCVs were also poorly susceptible to Cip. In contrast, the combination of Def-GaPP-Cip showed synergistic effects (Figure 4) resulting in significantly higher antibiofilm activity, even when treatment with the individual compounds was unsuccessful (Figure 3, SCV2). This confirms that the combination of drugs with different modes of action can be effective in treating bacteria with poor antibiotic susceptibility. The higher efficacy of the triple combinations Def-GaPP-Cip and Def-GaPP-Gent might rely on the synergy between reactive oxygen species (resulting from the Def-GaPP treatment) and antibiotics, as described elsewhere (Garcia L. G. et al., 2012; Garcia et al., 2013).
The antibiofilm activity of Def-GaPP was furthermore determined in an in vitro wound model where biofilms were grown on an artificial dermis and exposed to loaded gels. In line with results obtained in the colony biofilm model, Def-GaPP gel showed substantial antibiofilm effects, though to a lower extent. This may be the result of a nutrient-rich environment with blood/heme as favorable iron source. Bacteria recognize the tetrapyrrole ring of heme and GaPP, but can distinguish between both compounds (Stojiljkovic et al., 1999; Moriwaki et al., 2011). Therefore, GaPP gel showed only minor antibiofilm activity, in contrast to the Def-GaPP combination. The Def-induced chelation may have deprived bacteria of nutrients and increased the uptake of GaPP as heme-mimicking agent for a substantial antibiofilm effect of this "Trojan Horse" compound.
In an in vivo infection model in the nematode C. elegans the antibacterial effect of Def-GaPP was assessed. It was observed that the survival rate of infected nematodes were strain-dependent (Figure 7). Literature described that hemin/menadione auxotrophic strains were less virulent than parent strains and thymidine auxotrophic strains (Sifri et al., 2006). In line with this the order of virulence in the present study was observed to be SCV1 (hemin/menadione auxotroph) being the least and SCV2 (thymidine auxotroph) being the most virulent strain (Figure 7). The auxotrophy type can influence the strain's susceptibility to antibacterial compounds. In the colony biofilm model SCV1 was more susceptible to GaPP and Def-GaPP than SCV2, which required the combination of Def-GaPP for a substantial antibiofilm effect. Consistent with these observations similar results were observed in vivo in the C. elegans model. While infected nematodes died over time due to bacterial colonization, biofilm formation and toxin production (Sifri et al., 2005), the exposure to Def-GaPP prolonged the lifespan of all infected nematodes (Figure 7) and reduced the bacterial burden per worm (Figure 8). Treatment with GaPP alone only increased the survival rate of SCV1 and P1 infected worms, but failed in SCV2 infected worms. Therefore, combining GaPP with Def appeared to be crucial for an elevated antibacterial activity against S. aureus SCVs of any auxotrophy type.
The present study confirmed strong antibacterial and antibiofilm properties of Def-GaPP against S. aureus SCVs in vitro and in vivo. When applied in a surgical hydrogel, Def-GaPP has potential to complement the gel's wound healing properties with antibacterial and antibiofilm effects and could serve as an alternative treatment for biofilm and SCV-related infections. Due to the risk of emerging antibiotic resistance, which is in particular associated with prolonged antibiotic treatment, the combination of drugs with different modes of action is advantageous. The combination of Def-GaPP with antibiotics may facilitate a multi-pronged approach to increase the treatment efficacy against otherwise antibiotic tolerant/resistant S. aureus SCVs. While the results of the current pilot study are encouraging, the broader applicability of Def-GaPP for the treatment of SCVs derived from other species is on the way to be validated.
Author Contributions
Conception and design of study: KR, NT, TC, and SV. Acquisition and analysis of data: KR, NT, GZ, and TC. Drafting of article and/or critical revision: KR, NT, TC, CP, PW, and SV. All authors approved the final article.
This work was supported by The Hospital Research Foundation, Woodville, Australia; the Department of Surgery, Otolaryngology Head and Neck Surgery, University of Adelaide, Adelaide, Australia; an Australian Government Research Training Program Scholarship and The National Health and Medical Research Council, Australia [grant number NHMRC: GNT1090898]. The funders had no role in study design, data collection, analysis and interpretation of data, or the decision to submit the work for publication. KR is grateful for having received the Bertha Sudholz Research Scholarship by the Florey Medical Research Foundation, Adelaide, Australia and the Trevor Prescott Memorial Scholarship by The Freemasons Foundation, Adelaide, Australia.
Conflict of Interest Statement
Competing financial interests: PW holds a patent on the chitosan-dextran hydrogel. PW and SV hold a patent application on the treatment combination of deferiprone and gallium-protoporphyrin. The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Ilse Vandecandelaere and Jolien Claeys (Laboratory of Pharmaceutical Microbiology, Ghent University, Gent, Belgium) are kindly acknowledged for their assistance.
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Baumert, N., von Eiff, C., Schaaff, F., Peters, G., Proctor, R. A., and Sahl, H.-G. (2002). Physiology and antibiotic susceptibility of Staphylococcus aureus small colony variants. Microb. Drug Resist. 8, 253–260. doi: 10.1089/10766290260469507
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Mitchell, G., Brouillette, E., Séguin, D. L., Asselin, A.-E., Jacob, C. L., and Malouin, F. (2010a). A role for sigma factor B in the emergence of Staphylococcus aureus small-colony variants and elevated biofilm production resulting from an exposure to aminoglycosides. Microb. Pathog. 48, 18–27. doi: 10.1016/j.micpath.2009.10.003
Mitchell, G., Séguin, D. L., Asselin, A.-E., Déziel, E., Cantin, A. M., Frost, E. H., et al. (2010b). Staphylococcus aureus sigma B-dependent emergence of small-colony variants and biofilm production following exposure to Pseudomonas aeruginosa 4-hydroxy-2-heptylquinoline-N-oxide. BMC Microbiol. 10:33. doi: 10.1186/1471-2180-10-33
Moriwaki, Y., Caaveiro, J. M., Tanaka, Y., Tsutsumi, H., Hamachi, I., and Tsumoto, K. (2011). Molecular basis of recognition of antibacterial porphyrins by heme-transporter IsdH-NEAT3 of Staphylococcus aureus. Biochemistry 50, 7311–7320. doi: 10.1021/bi200493h
Morones-Ramirez, J. R., Winkler, J. A., Spina, C. S., and Collins, J. J. (2013). Silver enhances antibiotic activity against gram-negative bacteria. Sci. Transl. Med. 5, 190ra181. doi: 10.1126/scitranslmed.3006276
Proctor, R. A., von Eiff, C., Kahl, B. C., Becker, K., McNamara, P., Herrmann, M., et al. (2006). Small colony variants: a pathogenic form of bacteria that facilitates persistent and recurrent infections. Nat. Rev. Microbiol. 4, 295–305. doi: 10.1038/nrmicro1384
Reniere, M. L., Torres, V. J., and Skaar, E. P. (2007). Intracellular metalloporphyrin metabolism in Staphylococcus aureus. Biometals 20, 333–345. doi: 10.1007/s10534-006-9032-0
Richter, K., Ramezanpour, M., Thomas, N., Prestidge, C. A., Wormald, P.-J., and Vreugde, S. (2016). Mind "De GaPP": in vitro efficacy of deferiprone and gallium-protoporphyrin against Staphylococcus aureus biofilms. Int. Forum Allergy Rhinol. 6, 737–743. doi: 10.1002/alr.21735
Richter, K., Van den Driessche, F., and Coenye, T. (2017a). Innovative approaches to treat Staphylococcus aureus biofilm-related infections. Essays Biochem. 61, 61–70. doi: 10.1042/EBC20160056
Richter, K., Thomas, N., Claeys, J., McGuane, J., Prestidge, C. A., Coenye, T., et al. (2017b). A topical hydrogel with deferiprone and gallium-protoporphyrin targets bacterial iron metabolism and has antibiofilm activity. Antimicrob. Agents Chemother. 61:00481–17. doi: doi: 10.1128/AAC.00481-17
Sifri, C. D., Baresch-Bernal, A., Calderwood, S. B., and von Eiff, C. (2006). Virulence of Staphylococcus aureus small colony variants in the Caenorhabditis elegans infection model. Infect. Immun. 74, 1091–1096. doi: 10.1128/IAI.74.2.1091-1096.2006
Sifri, C. D., Begun, J., and Ausubel, F. M. (2005). The worm has turned–microbial virulence modeled in Caenorhabditis elegans. Trends Microbiol. 13, 119–127. doi: 10.1016/j.tim.2005.01.003
Skaar, E. P. (2010). The battle for iron between bacterial pathogens and their vertebrate hosts. PLoS Pathog. 6:e1000949. doi: 10.1371/journal.ppat.1000949
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Stojiljkovic, I., Evavold, B. D., and Kumar, V. (2001). Antimicrobial properties of porphyrins. Expert Opin. Invest. Drugs 10, 309–320. doi: 10.1517/13543722.214.171.1249
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Tan, N. W., Cooksley, C., Roscioli, E., Drilling, A., Douglas, R., Wormald, P. J., et al. (2014). Small-colony variants and phenotype switching of intracellular Staphylococcus aureus in chronic rhinosinusitis. Allergy 69, 1364–1371. doi: 10.1111/all.12457
Thangamani, S., Younis, W., and Seleem, M. N. (2015). Repurposing celecoxib as a topical antimicrobial agent. Front. Microbiol. 6:750. doi: 10.3389/fmicb.2015.00750
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von Eiff, C. (2008). Staphylococcus aureus small colony variants: a challenge to microbiologists and clinicians. Int. J. Antimicrob. Agents 31, 507–510. doi: 10.1016/j.ijantimicag.2007.10.026
von Eiff, C., Bettin, D., Proctor, R. A., Rolauffs, B., Lindner, N., Winkelmann, W., et al. (1997a). Recovery of small colony variants of Streptococcus aureus following gentamicin bead placement for osteomyelitis. Clin. Infect. Dis. 25, 1250–1251. doi: 10.1086/516962
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Weinberg, E. D. (2009). Iron availability and infection. Biochim. Biophys. Acta 1790, 600–605. doi: 10.1016/j.bbagen.2008.07.002
Wiegand, I., Hilpert, K., and Hancock, R. E. (2008). Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat. Protoc. 3, 163–175. doi: 10.1038/nprot.2007.521
Keywords: biofilms, small colony variants, Staphylococcus aureus, iron, deferiprone, gallium-protoporphyrin, wound models, Caenorhabditis elegans
Citation: Richter K, Thomas N, Zhang G, Prestidge CA, Coenye T, Wormald P-J and Vreugde S (2017) Deferiprone and Gallium-Protoporphyrin Have the Capacity to Potentiate the Activity of Antibiotics in Staphylococcus aureus Small Colony Variants. Front. Cell. Infect. Microbiol. 7:280. doi: 10.3389/fcimb.2017.00280
Received: 26 April 2017; Accepted: 08 June 2017;
Published: 22 June 2017.
Edited by:Yinduo Ji, University of Minnesota Twin Cities, United States
Reviewed by:Chia Y. Lee, University of Arkansas for Medical Sciences, United States
Mark Smeltzer, University of Arkansas for Medical Sciences, United States
Copyright © 2017 Richter, Thomas, Zhang, Prestidge, Coenye, Wormald and Vreugde. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Sarah Vreugde, email@example.com


text:
Foraging, infusing, shaking, stirring: this one's got it all. How do you make weird, fancy drinks at home? How much booze should you buy for a party? What drinks do bartenders hate to make? How can you avoid a hangover? What are the best mocktails? Famed bar chef and mixologist Matthew Biancaniello tours Alie through Malibu fire aftermath and talks about his boggling backstory, why he doesn't go out much, the secret formula to making good drinks and why you shouldn't be afraid to be weird.
Matthew's website and Instagram @EatYourDrink
More links at www.alieward.com
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Sound editing by Steven Ray Morris and Jarrett Sleeper
Theme song by Nick Thorburn
Support the show: http://Patreon.com/ologies
See omnystudio.com/policies/listener for privacy information.

subset: commoncrawl


text:
Sample Sales Assistance CV Cover Letter Template
Ms. Kirsten Carter
HR Manager
UK Town Mall
Dear Ms. Carter:
I have read at your company website that you are currently in need of sales assistants to be assigned in some stores of your mall. I believe I have the abilities that suit the required qualifications for this job post.
I have worked as a sale assistant for other companies in the past, and I believe I have more than enough training to please the mall's customers. In spite of this, I am still willing to undergo training if your company requires it. I am sending you a copy of my detailed CV for your perusal. In the CV are some character references to which you can verify my work experience and ethics.
I am willing to attend an interview regarding this job application at your most convenient time.
I look forward to hearing from you.
Thank you and more power.
Sincerely yours,
Kate Hall


text:
A thermocouple is a circuit that consists of
two different wires or alloys, welded in their ends, and an
electromotive force (E.M.F.) appears between them due to the
effect of temperature.
A thermocouple allows knowing the temperature
in its union T1 by connecting it to a measure instrument.
Thermocouples are used as probes in control
or regulation measures of temperature in materials put under
heat treatements.
There are several kinds of thermocouples, depending on wire composition,
that also depends on the temperature to measure and its exposition
time. Previous section supedites the diameter of wires to use.
Insertion thermocouple>>
<< Sheathed thermocouple


text:
Who Is Hillary Clinton's Makeup Artist?
Makeup is no effing joke — it's the difference between looking hungover and fresh-faced on a Friday after a rather wild night out for Thirsty Thursday. More importantly, it has the power to affect how people perceive your overall health and even personality. Hillary Clinton's makeup artist Barbara Lacy will do just that, as Clinton begins her campaign for presidency.
So who is Barbara Lacy, exactly? Well, to start, she's been our former First Lady's makeup artist for over a decade now. She has also served as the makeup artist for the comedy TV show Veep, although Lacy herself doesn't work on Julia Louis-Dreyfus' makeup on set. Aside from her White House work, she's got a pretty impressive resume, serving as the makeup artist for movies and TV shows, including Transformers, Step Up and The West Wing.
And this isn't the only time she's helped Clinton through her presidential campaign. Back in 2008, Lacy was in charge of how Clinton looked in interviews and appearances, and told the Hill in 2010, "If you don't look healthy, if you look sickly or tired, these are things that people judge you by if they're just looking at you and not listening to you."
Lacy continued on, telling the Hill, "My whole job was to make sure that people weren't focused on [Clinton's] face. Her face can't be a distraction. And that means just looking good and healthy and rested. You're noticing that the person really looks good, and you're not really sure why, so you keep staring at them. And that's the art."
In addition to building up her image, Clinton has also taken Kristina Schake as part of her communications team to avoid a mistake that "dodged" her the 2008 campaign—showing her softer side, according to The New York Times. This super star team that Clinton has garnered together, Lacy included, are bringing out the best possible Hillary they can. And tbh, they're pretty much already there. So sorry Veep, you'll probably be receiving a group text from Hillary later.
Images: Getty


text:
Twitter Us
Friend Us
Inflatable Pirate Ship Cooler is a fun way to serve your cold drinks. Cooler is made of plastic and measures approximately 41" wide x 23" deep x 15" high. Pirate Ship holds 72 - 12 oz. Cans (not included). Great for indoor and outdoor parties. Warning: Choking Hazard - Small Parts. Not for children under 3 years. This item is not designed to be used as a flotation device. Look for other pirate party supplies (sold separately).
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Halloween occurs at the time of the year when the last of the harvest is being gathered, the days are cooler, and the nights come earlier and seem more mysterious. Its a time for children of all ages to enjoy the preparations as they invite witches and monsters and hobgoblins home.
"Games, music, costumes, party supplies -- we can come to you or you can come to us."
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text:
Question: Is it true that 7th Heaven is back for a full, 22-episode season?
Answer: Yes, which is strange since I'm pretty sure the CW originally picked it up for only 13. It'd be nice if just once the CW made a decision that resulted in fewer 7th Heaven episodes, rather than more. Is that too much to ask?


text:
Missed the price on this one..
5.10 Catterick - Young Tiger e/w 9/1
Missed the price on this one..
5.10 Catterick - Young Tiger e/w 9/1
4.35 Laytown - Caesar's Comet e/w 15/2
2.10 Doncaster - Keep Busy e/w 18/1
Last edited by typhoon; 11-09-2019 at 22:15.
2.20 Sandown - Sand Diego 9/2
Well done typhoon
why does it hurt when I pee ? Frank Zappa R.I.P
4.55 Kempton - Parkers Boy e/w 20/1
But keep it up mate, i'm already looking forward to the next one. And i shall watch the race with added interest.
a rather simplistic Analysis but it works out sometimes, the problem is I can often beat the Bookie's odds but getting them to win is a different matter altogether, if we get market support 15 minutes before the race then I know I'm not far off.
Last edited by typhoon; 16-09-2019 at 13:39.
unplaced on to tomorrow..
2.30 Redcar -[B]
Last edited by typhoon; 17-09-2019 at 14:39.
At all points in our lives you will either be underrated or overrated
not sure what happened there, somehow I edited and replied at same time, anyway the selection Rusalka was 3rd
Cracking call mate.
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text:
Visualized data chart design, simple and intuitive statistical charts have made it clear the business situation and change of each store.
The multi-dimensional analysis of business data makes it easy to objectively and accurately grasp the business conditions of the enterprise, understand market demand, and create greater benefits.
Combine KPI data, which makes it possible to understand store operations and product sales in real time, and let it be easy to make timely decisions on product sales and store management
Key point sharing, mark and share the key point of the interface data, and the subordinates will more clearly understand the opinions of the superior leaders.
Help the shopping guide to keep track of the performance balance, achievement rate of performance and ranking change, in order to have a fully understanding of her/his own ability.
Classified membership management (birthday, new member, dormant member), which facilitate the communication between the shopping guide and the member
Making the shopping guide feeling at home, skillful in sharing experience and learning techniques
Easy for sharing goods, can operate with mobile phone and sale online, and know who bought it and who watched it.
Can respond to customers at any time, and provide customers with considerate product maintenance and product consulting services to improve brand affinity, to and be a never-offline service provider.
采用最先进的 spring框架+ice服务开发的一套系统
进行会员招募, 会员运营,使微商城进行线上反哺线下实现
The most advanced spring framework+a series of system
developed by ice service Full function, easy to operate,high
security, cooperate with Bizvane members to recruit new
members through various activities, by the member business
to achieve online feedback and offline sales increase
Manage multiple brand official accounts at the same time
Personalized decoration Wechat-E-shop, adjust price to local conditions
Simple and flexible member marketing management
Online and offline data integration
BIZVANE WeChat operation service is based on the original service format of BIZVANE, which focus on providing customers with software-oriented, sales-oriented WeChat channel operation services. The content includes: WeChat operation, Wechat E-shop operation service, WeChat operation is based on the official account of the customer, which aims at planning activities for customers, Write articles and design pictures, using the corporate public number to increase the influence of corporate brand communication, increasing the number of corporate public fans, and ultimately Get massive membership data and combine our other products for accurate CRM operations.
Our team is familiar with various Wechat E-shop products in the market, such as Youzan, Weimob, Renrenshop, etc., which can assist and guide customers to operate the micro-sales according to the individual needs of different customers. We can also provide Wechat-E-shop precise operation services for customers who use our Wechat-E-shop tools, including member recruitment, event planning, data analysis and so on.


text: I ran across the Museum of Menstruation and Women's Health the other day when I was looking for Gloria Steinem's 1978 essay, "If Men Could Menstruate." The essay is worth looking up, but it turns out so is this web site. The founder is a man, Harry Finley (whose bio is here) and it's not exactly clear what influenced him to set up this web site, but I'm glad something did, because this site is a treasure trove of information about a subject that every woman obsesses about, but often knows very little about. And let's not even get into how well most men understand menstruation, let alone empathize with us about it.


text:
New hope for treating muscular dystrophy
Researchers tout new technique with gene editing
After decades of disappointingly slow progress, researchers have taken a substantial step toward a possible treatment for Duchenne muscular dystrophy with the help of a powerful new gene-editing technique.
Duchenne muscular dystrophy is a progressive muscle-wasting disease that affects boys, putting them in wheelchairs by age 10, followed by an early death from heart failure or breathing difficulties.
Hereditary illness
The disease is caused by defects in a gene that encodes a protein called dystrophin, which is essential for proper muscle function.
Because the disease is devastating and incurable, and common for a hereditary illness, it has long been a target for gene therapy, though without success.
An alternative treatment, drugs based on chemicals known as antisense oligonucleotides, is now in clinical trials.
But gene therapy - the idea of curing a genetic disease by inserting the correct gene into damaged cells - is making a comeback.
A new technique, known as Crispr-Cas9, lets researchers cut the DNA of chromosomes at selected sites to remove segments or insert new ones.
Three research groups, working independently of one another, reported in the journal Science on Thursday that they had used the Crispr-Cas9 technique to treat mice with a defective dystrophin gene.
Tests with mice
Each group loaded the DNA-cutting system onto a virus that infected the mice's muscle cells and excised from the gene a defective stretch of DNA known as an exon.
Without the defective exon, the muscle cells made a shortened dystrophin protein that was nonetheless functional, giving all of the mice more strength.
The teams were led by Charles Gersbach of Duke University, Eric Olson of the University of Texas Southwestern Medical Center in Dallas and Amy Wagers of Harvard University.
"The papers are pretty significant," said Louis Kunkel, a muscular dystrophy expert at Boston Children's Hospital who discovered the dystrophin gene in 1986.
All three teams have filed for patents.
But considerable work lies ahead before clinical trials can start. It is not clear how the human immune system would react to the components of the gene-editing system or to modified dystrophin proteins to which it has not been habituated.


text:
DSS 2000 series Dust Separation System
Dedicated designs to separate filler/dust from small size aggregates and deliver much improved aggregate where there is a high filler component, according to specifications. The systems are very easy to locate on site utilizing relatively little space, and simply have to be positioned at the outlet of the incoming aggregate conveyor with provision for the outward conveying of the processed aggregates.
DSS 2000 series systems comprise three main sub-systems, namely:
- Blower – to generate system air velocity
- Tumbler – a special low speed rotating agitator for separating dust from aggregate
- Cyclone system – a twin system for both coarse and fine separation
- Best dust separation
- Very low wear and maintenance
- High efficiency
- Capacity from 30 to 100 tons per hour depending on material humidity and particle size (my instinct is to cut this as it should be assumed knowledge)
- Filler percentage from variable and average 5 to 8% depending on material
- Screw conveyor dust dischargers
- Convenient low inlet height
- Plant has overall low profile
- High recirculation ratio giving most efficient air use
- Very good discharge air quality to meet environmental demands
- Low operating costs


text:
Norwegian leased the airplane through an agreement with MG Aviation.
Norwegian already operates eight 787-8s and will use its 787-9s to open up new routes across North America, South America and Southeast Asia with its increased range and capacity.
The 787-9 complements and extends the super-efficient 787 family. With the fuselage stretched by 20 feet (6 meters) over the 787-8, the 787-9 will fly more passengers and more cargo farther with the same exceptional environmental performance — 20 percent less fuel use and 20 percent fewer emissions than similarly sized airplanes.
Norwegian serves more than 130 destinations across Europe, North Africa, the Middle East, the USA and Southeast Asia, with a fleet that includes 90 Next-Generation 737-800s and eight 787-8s. In October 2015 the airline placed the largest single order for 787-9s from a European carrier, ordering 19 airplanes valued at more than $5 billion at list prices.
Source: airlinersphotostream.com


text:
Final chapter for struggling Dymocks
After a 10-year stint in Albany, Dymocks will close in December, joining many other local businesses which have shut their doors in the last year.
Owner Deborah Congdon established the business with her late husband in 2008.
She said in the last year times had been tough, struggling with the high costs of rent and keeping up with online sales.
"The rent is too high, it's ridiculous," she said. "It is a financial struggle to keep up with the rent, and there isn't anything we can do apart from close our doors."
Ms Congdon said more and more customers would opt for an online purchase rather than waiting the two weeks to order a book in.
"To survive you have to be cheap and compete with online prices, you have to get the books in fast and you have to keep up with the ridiculous rent, and lately costs have been going up and up and book sales have been going down," she said.
The business owner said the future of book shops in Albany is worrying. "I think in Australia it will be fine but in Albany it won't survive, and that's really sad," she said.
"I am very sad to close doors, I have cried many days.
"It's been emotional but nothing can be done."
Dymocks is among other businesses to have closed their doors recently, including Bravo Shoes, Albany Surf and Skate, Movie HQ, Southern Stationery, Bush Fairy and Just Add Ice.
Ms Congdon said she would like to see a change in the local council.
"There needs to be more support, more guidance and more communication," she said.
"This end of York Street is struggling and we aren't getting any help or support. It would have been nice to have received a flyer before they organised some Perth mystery shopper to pick us apart.
"If the council don't step up and change, I think there will be a lot more businesses closing their doors and it's very sad.
"I worry for the future of local business on York Street."
Dymocks will close its doors on December 6.
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Fifth in a series
My wife is a Certified Registered Nurse Anesthetist (CRNA) for a local hospital, so she puts people under sedation before they go into surgery.
I sometimes joke that she puts people to sleep Monday through Friday, and as a pastor I put people to sleep only on Sundays and Wednesdays.
Often, when others find out she is an anesthetist, they wonder aloud that it must be tricky to put someone to sleep. My wife usually responds that putting someone under is not the trick – it's waking them back up!
As I was reading about the church at Sardis in Revelation 3:1-6, these same conversations kept coming back to my mind within the context of the local church.
The Lord warns the church at Sardis against being alive in name only, while they were truly lifeless and dead. Jesus issues a call for the church to come alive and be watchful.
It is apparent the church at Sardis was once great, because they had a name of being alive.
This sounds like so many churches in modern day America, still living in the glory days of what happened when our grandparents were in church. "Wake up, be watchful, and repent!" Jesus says to us.
Ancient Sardis was known for its rich history, military defense and wealth, but the church had grown complacent and had been lulled to sleep without even realizing what happened.
Jesus' command to "wake up and be watchful" was an indictment against the church because He "did not find their works perfect before God."
We always look for someone to blame, so whose fault was it that this church was sleeping on the job? Was it the pastor's fault, or maybe the deacons or better yet the music leaders? As the church is close to death, Jesus gives a warning that "If you will not watch, I will come upon you as a thief, and you will not know the hour I will come upon you!" He calls each of them to repentance, or risk facing His judgement.
Jesus acknowledged there were a few dedicated people still holding on in a dying church. There was something very different about them, because they had not defiled their garments. The true believers were known to be worshippers that did not compromise nor grow comfortable.
Christians, be encouraged. There is no person or congregation beyond the hope, help and healing of Jesus. So, church, be watchful!
Related columns:
Ephesus: Have we lost our first love?
Smyrna: The poor church that was rich
Pergamum: No compromises
Thyatira: a call to church discipline
(EDITOR'S NOTE – This article is part of a series on the theme of the 2018 North Carolina Pastors' Conference, "7 Churches of Revelation." This year's event will occur Nov. 4-5 in conjunction with the Baptist State Convention of North Carolina's annual meeting. Visit ncpastorsconference.org for more information. Chip Hannah is senior pastor of Peace Baptist Church in Whiteville. Each column in the series is written by a different N.C. leader and refers to one of the seven churches in Revelation.)


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No Monkeying Around for New Chinese Tea Start-up Targeting China's Smaller Cities
Co-founded by former 7th Grand Slam winner, a new tea start-up is taking China's smaller cities by storm.
New Chinese tea brand, Reinforcements of the Monkey (猴子的救兵), opened 100 stores in 17 days in China's small and medium-sized cities, according to Financial Circle. The new Chinese tea start-up beat HeyTea's past plan to open 100 new stores in China, Singapore, and Hong Kong by the end of 2018.
In Mar 2019, the new tea brand cooperated with a Chinese business platform, "Where is the Business Opportunity" to achieve its goal.
With tea brands such as Nayuki and HeyTea fought in China's tier one cities, Reinforcements of the Monkey focused on smaller towns, seeing success with its differentiated positioning and cost-effective business route.
Unlike its first-tier competitors, one store is approximately 15 square meters and operated by two people. The overall cost of opening one store is less than CNY 100,000.
The company's co-founder, ZHANG Jike (张继科), former ping-pong winner of the 7th Grand Slam, realized how China's smaller cities was an underserved tea community. The market scale of new tea market development exceeded CNY 90 billion in 2018, and brands such as Nayuki had already proven their profitable business models in first-tier cities.
Reinforcements of the Monkey officially launched in Mar 2019; its minimalist appearance with black and white tones became the tea market's dark horse. After six months of research and development, the new tea brand launched a series of products catered to suit the taste of different target demographics.
A distinct advantage the Chinese tea start-up has over other tea brands is powerful endorsements. The brand became powerful due to not only ZHANG Jike's fame but also support from his celebrity friend circle such as ping pong champions WANG Hao (王皓) and KONG Linghui (孔令辉); badminton champion, CAI Wei (蔡赟), and film stars MA Su (马苏), DOU Xiao (窦骁), DONG Qing (董晴), and more.
Also, it brings what one would expect to see and feel from new tea start-ups in China's first-tier cities to unsatisfied markets. According to Jiguang, start-ups can usually monopolize the market they began in. Therefore, this gives it a competitive advantage. However, there's still ample room for growth in providing convenience for consumers by ordering online or eventually through its app. How the company decides to expand in its downward supply chain will be critical in the future.
HeyTea's advantages lie in its strong network effect, effective WOM (word of mouth) marketing, and customer convenience. Along with Nayuki, it opened a tea room to arguably compete with the atmosphere Starbucks provides. While it's too early to tell whether Reinforcements of the Monkey will venture into first-tier cities, it may stand a good chance in the future due to its reduced marketing costs. According to the South China Morning Post, HeyTea "[saved] millions in marketing" relying on WOM.
The company hopes to become a low-cost entrepreneurial brand with stable profitability. Reinforcements of the Monkey began its operations in Aug 2018 and had a registered capital of CNY 116.1 million.


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The West Australian Good Food Guide 2015 is your definitive guide to dining out in WA's restaurants, bars and cafes for only AU$10.99.
If there were a theme to the 2014-15 culinary calendar, it would be the rise of the small bar as food leader. No-one could have foreseen this when small bar licences were passed into law by the WA Parliament eight years ago. But much of what is interesting and innovative in food isn't coming from the big name white-glove establishments, it's flowing forth from the unfettered creativity of our small bar chefs. While not a new phenomenon in other parts of Australia, Perth stands alone in its food leadership in bars. In Sydney and Melbourne, for instance, the cutting edge of food is still mainly evolving in restaurants, not bars. In Perth, Darlings Supper Club, Lot Twenty, Pleased To Meet You, Swings Taphouse, Old Faithful Bar & BBQ, Morries Anytime and The Cabin are rocking a new aesthetic in accessible, fun, share-style food with bold flavours and brave flavour combinations. Much of it is the sort of food chefs cook for each other – so-called dude food. And although we're wary at the Good Food Guide about celebrating food fads and fleeting fashion, the 'dudes' may well have left an indelible mark on our local, home-grown cuisine, for which we can all be thankful. Enjoy the Guide and happy eating!


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Unlike some people who were drawn to science, I am not a seriously math-oriented person. Sure, I took the algebra and calculus required to obtain my Bachelor of Science degree, but I didn't feel the need to be doing calculations of my own on a daily basis. This was one of the reasons which drew me into Geology—it is a science, but there are many disciplines in geology wherein math isn't needed often. For my Master's degree the only calculations I needed to make were all done in a single afternoon. I needed to convert from feet/miles to meters/kilometers since my base map was in feet (my advisor and I both felt that metric is the more appropriate system in which to report results). This conversion is taught in pretty much all first-year geology laboratory courses in the US, and not only had I taken such a class, I was teaching that class that year, so I had the formula immediately to hand. I also needed to calculate apparent dip (what angle the tilted layer of a fold in the rock will appear to have if you happen to slice through it at some angle other than exactly perpendicular to it). This is taught in structural geology laboratory courses, and again, I happened to be teaching that class that semester, so had the necessary formulas at the ready.
For my PhD project, I am needing to do a fair few more calculations, on a much more regular basis. However, I don't actually do the arithmetic myself; instead I set up Excel Spreadsheets with formulas as I need them, and then type a number into the appropriate cell, and read the answer from another cell. This is a very, very useful skill to have. It requires care to get the formulas typed in correctly, and sufficient understanding of what you want them to do and what sort of answer you should get so as to be able to tell if you've got it right and it is working, but once it is done correctly it can be re-used any number of times by just changing the contents of the input cell and reading the answer out of the output cell.
Today I used my spreadsheet formula building skill for something unrelated to my PhD project. For the past year I have been working on and off on making a hat by nålbinding. This has been my project which stays in my backpack in case I happen to be stuck in line somewhere, or attending a lecture, or otherwise have some time with nothing I need to be doing with my hands. A few people have asked me how long it has taken me, and while I know the date I started the project (18 May, 2008), I didn't have a clue how much time has been spent working on it. So I set up a spreadsheet to figure it out.
I first looked up the formula to calculate the circumference of a circle (2μr) to be certain I had remembered it correctly from primary school (I had). I then told Excel to put the value for μ in cell A1 (one does this by typing "=PI()" into that cell). I then typed the headings "diameter", "radius" and "circumference" into cells B1, C1, and D1. In the next row I set up my formulas. I measured the diameter of the hat (42 cm at the widest point thus far) and entered it into the empty cell in column B. In the adjacent cell I typed "=B2/2", which told the computer to calculate the radius by dividing the diameter by two. In the third cell I typed "=2*$A$1*C4", which told it to calculate the circumference by multiplying two times the value in A1 (which, as you recall, is Pi), times the radius. For the outside row of the hat this is 129.6 cm. Checking that with my tape measure confirms that this is pretty much correct (keeping in mind that the hat, being made of yarn, is able to stretch a bit, so obtaining an accurate measurement can be tricky).
(It is important to remember that in Excel, if you copy/paste a formula and the formula contains a cell name, the pasted version will instead name of the cell which is located in the same position relative to the new cell as the originally named cell is in relation to the original formula location. This is useful when you want to perform the same calculation over and over again using different starting data. However, sometimes you want the formula to point to a specific cell no matter what. In that case you add the $ symbol to the name of the cell. This is why the above formula contains both cell names with and without the $ symbol—I needed them in the name of the cell containing the value for Pi, because I wanted to use that particular value every time I calculated the circumference, but I didn't want them in the name of cell containing the radius, because I knew that I would soon need to be calculating the circumference for lots of different radiuses.)
Having set up the first step, it was time to do the real calculations; if I want to know how long it took to make the hat, I need to know how many knots it took to make it. This hat is constructed by tying knots in a spiral pattern, starting from the center, and working outwards in what could be thought of as one continuous row, or could be thought of as a bunch of rows, each one out from the next. The latter interpretation is more useful for my purposes today, so first I counted the number of rows from the center to the outside (75). Then I measured the diameter of the innermost loop of that spiral (0.8 cm) and typed it into the 75th row of the spreadsheet in column B. I then entered "=B2-B75" into random cell out of the way of where I was working, and then (in cell F75) divided that result by 74 to get the width of a typical row (if there are 75 rows then there are only 74 spaces between them, which is why I used 74 and not 75 in that formula—if you don't believe me, look at your hand and count the number of fingers and compare that total with the number of spaces between them). I then typed "=B2-$F$75" into cell B3, and then copied that formula into all of the rows between that cell and cell B75. This gave me the diameter of each row. I then copied the formulas for radius and circumference into all of those in between rows as well, giving me 75 rows worth of steadily decreasing circumference totals. From there it was a simple matter to add them all up (not quite 6000 cm worth of total distance for an ant to crawl if it started in the center and followed the spiral out to the edge), and multiply by the number of knots per cm (about 5), giving me a grand total of almost 27,800 knots in the hat. I then set my stopwatch running and made a few knots, and determined that if all goes well and there are no tangles in the yarn, I can do a knot in about 10 seconds, more or less. Assuming that this rate has been typical throughout the project, the computer tells me that I've put in about 77 hours work on this hat, which averages to about 1.7 hours a week (or less than 15 minutes a day) since I started it. (Keep in mind that some weeks it would not have been touched at all!) All in all, this sounds like a reasonable amount of time, particularly since it mostly exists as a project to keep me from being bored when I'm waiting for something.
So, the next time you hear a student complaining about their math class that they will "never need this stuff", you can assure them for me that, yes, actually, it does come in handy, sometimes in very unexpected ways.


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A long, long time ago...
I can still remember
How that music used to make me smile.
And I knew if I had my chance
That I could make those people dance
And, maybe, theyd be happy for a while…
So bye-bye Miss. American Pie. It was the dawning of the Age of Aquarius, Woodstock, and the summer of love, mini skirts and bell-bottoms. On TV, the Beaver and Wally were out. Pete, Julie and Linc --The Mod Squad were in. (Or so I am told. I am much too young to remember myself).
In episode 31 Linc was in a relationship with a secretive woman. The affair seemed doomed from the start. The fetching woman had a young child. This leads me to believe that she may have been a mommy blogger except that mommy bloggers have no secrets and it was 1969, so probably not.
What does this have to do with today's post? Nothing really, except for the Linc part. If Linc were Link then this would be totally relevant and you would immediately understand what I am saying. As for the Don Mclean lyrics? Well, that is because I like pie and Miss America...Today's post is about links, pie, and Miss America. Make sense now?
I love links and fetching women. In that spirit, here are links to some posts I have recently read and loved. Any one of these chicks could have been Julie on The Mod Squad. They are just that cool.
3.mackin ink
4.the bloggess
5.desire to inspire
*If you are reading my list of links and saying to yourself "What the hell is wrong with her? These people are A list bloggers and everyone knows who they are. I saw these posts days ago...GAH!" I would like to say that I am not cool enough to be Julie, they are. Additionally I only discovered Google reader a couple of weeks ago (seriously). May I politely ask why you are holding out on me? If there are, blogs that you love please share.
Debra Barnes
Miss. America 1968
She was from Kansas-- She was also the first Miss. America to get her ass kicked by pissed off feminists (figuratively of course).
Alas, I have given up my dream of being Miss. America. I was never really cut out for the job. My measurements are all wrong. The talent portion of the competition would have been a problem. Then there is the whole "nice" thing. I think I would have made a better protester. Maybe it is not too late for that.
1.Cake or Pie?
Answer: Pie
2. Does Big Judes read your blog? Does she know you have a blog?
Answer: See above.
3. Do you adore valances, swags, scarves?
4.Is your lawn everything it could be and more?
5. Have you changed your HVAC filters within the past 6 months?
Answer: Pie
6.Did you ever wear pegged-leg jeans or tight-rolled jeans? Are there photos?
Answer: I am a fan of the Boyfriend jean. Cuffed jeans if worn correctly can be good. Do not use Katie Holmes as an example. I do have pictures. Is she not the cutest thing you have ever seen? Obviously there is not a lot of excess pie consumption going on. I admire her restraint and her excellent sense of style.
Answer: If you are referring to pie –Yes! If you mean Coffee and Cigarettes? Occasionally. If you mean beer, Yak.


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Sniffany Mat
Designer shopping bags and gift boxes generally don't make good beds, until now!
This luxury mat features a parody design of a popular brand's gift box. Complete with logo embroidery and the brand's signature color for that little bit of fashionista flare. Very plush and comfy!
Available in one size and style.
*Online prices may not reflect in-store pricing*


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A few parish unions cross county boundaries
HC means Holy Communion
MP means Morning Prayer
St. Canice's Cathedral, Kilkenny: Sunday 11.30 a.m.
and Wednesday 10.30 a.m.
St John's Church, Kilkenny: Sunday 10.00 a.m.
St Lachtain's Church, Freshford: Sunday 10.00 a.m.
St Aidan's Church, Kilmanagh: Sunday 10.00 a.m.
These are the usual service times but there are special holiday and united services
Dean: The Very Reverend David McDonnell
Curate: The Reverend Dr Richard Marsh
1st Sunday
Kells Church: 10.00 a.m.
Inistioge Church: 11.45 a.m.
2nd Sunday
Kilmoganny Church: 10.00 a.m.
Kilfane Church: 11.45 a.m.
Inistioge Church: 11.45. a.m.
3rd Sunday
Ennisnag Church: 10.00 a.m.
Inistioge Church: 11.45 a.m.
4th Sunday
Kilmoganny Church: 10.00 a.m.
Kilfane Church: 11.45 a.m.
5th Sunday
Kells Church: (combined service) 11.00 a.m.
Rector:The Reverend Canon James Mulhall
FIDDOWN UNION (1 church – St Paul's, Piltown – is in County Kilkenny)
Saint Paul's Church, Piltown:
1st Sunday 9.30 a.m. (MP)
2nd Sunday 9.30 a.m. (HC)
3rd Sunday 11.00 a.m. (MP) or All Age Worship
4th Sunday 11.00 (HC)
Holy Trinity, Portlaw and Saint Mary's, Kilmeaden:
1st Sunday 11.00 a.m. Holy Communion, Holy Trinity, Portlaw
2nd Sunday 11.00 a.m. (MP) Saint Mary's, Kilmeaden
3rd Sunday 9:30 a.m. (HC) Holy Trinity, Portlaw
4th Sunday 9.30 a.m. (HC) Saint Mary's, Kilmeaden
5th Sunday 11.00am Fiddown Union United Service
The three churches take turns in providing the location.
Rector: The Reverend Vicki Lynch
Also see:
KILCOOLEY UNION of Parishes WITH FERTAGH (1 church – Fertagh (Johnstown) – is in County Kilkenny
From end November 2019:
Sunday 1st: Crohane 9.00 (HC*); Kilcooley 9.00 (MP*); Littleton 10.15 (MP); Johnstown 11.30 (HC)
Sunday 8th: Littleton 9.00 (MP); Crohane 10.15 (MP); Johnstown 11.30 (MP); Kilcooley 11.30 (HC).
Sunday 15th: Crohane 9.00 (MP); Littleton 10.15 (HC*); Kilcooley 10.15 (MP); Johnstown 11.30 (HC*)
Sunday 22nd: 11 a.m. Crohane Carol service for Kilcooley Union; Kilcooley 11.30 (HC*); Johnstown 7.30 p.m. Carol Service.
Rector: The Reverend Graham Sawyer
(The monthly Sunday evening service at Kilcooley is permanently replaced by a morning service. The Sunday morning services at Johnstown will be at 11.30 from the beginning of December. Those services marked with an * are in traditional language following the principle of providing different styles and rhythms of worship at different times).
Rector: The Reverend Graham Sawyer
View Dioceses of Cashel, Ferns & Ossory in a larger map


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Jules Zacher, Esq. is currently representing a patient of Miami Valley Hospital who contracted Legionnaires' disease during his stay in the hospital's new patient tower. As of March 11, 2011, 10 patients of Miami Valley have become ill with Legionnaires' disease and one has become ill with Pontiac Fever, a milder strain of legionellosis. So far, the following information can be confirmed.
1. On December 28, 2010, Miami Valley Hospital began moving patients into the new patient tower, which houses the Heart and Vascular Center. At an undetermined time before the tower was opened to patients, the hospital flushed the plumbing of the addition, which is proper procedure following construction.
2. On February 3, 2011, the first case of Legionnaires' disease was confirmed.
3. On February 17, 2011, the second case of Legionnaires' disease was confirmed.
4. On February 19, 2011, the third case of Legionnaires' disease was confirmed.
5. On February 20, 2011, the fourth case of Legionnaires' disease was confirmed.
6. On February 22, 2011, the hospital implemented water restrictions and a program of disinfection involving hyper-chlorination of super-heating of the water system in the new patient tower.
7. On February 25, 2011, Miami Valley Hospital's water was declared safe.
It is not uncommon for cases of Legionnaires' disease to follow episodes of major construction. Legionellae bacteria occur naturally in the environment, so when construction breaks up soil containing legionellae, the disturbed bacteria can settle in the new building's water system. Legionellae thrive in areas of warm, stagnant water, and are fed by the dust and dirt of a construction site. It is essential, therefore, to flush the plumbing of a new building in order to thoroughly remove dirt and bacteria that may have settled there.
Miami Valley apparently completed this important step, yet legionellae still found their way into the new patient tower's water system, resulting in an outbreak of Legionnaires' disease among several patients. Not every case was diagnosed at the same time, however, as the timeline above demonstrates. The first case of Legionnaires' disease was confirmed on February 3, 2011; at this point, the hospital should have acted immediately to curb patients' potential exposure to bacteria in the water system and thereby prevent additional cases. But the hospital waited until three more cases were confirmed – three weeks later – before it restricted water use and disinfected the system. If Miami Valley had responded more quickly and attentively to the first diagnosis, 10 of its patients would not have been infected with the bacteria that causes Legionnaires' disease.


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We completed the Lake Superior Circle tour the first week of August. We left from Kalamazoo, Michigan, on Saturday July 30th and headed to Sault Ste. Marie, Michigan, with four other riders.
Saturday night we stayed at Brimley State Park. Sunday morning Frank and I left the other four and headed for Copper Harbor, Michigan, via Whitefish Point. We didn't make it that far the first day. We stayed in a state park again the second night. Monday we rode through Houghton/Hancock up to Copper Harbor and back down into Wisconsin towards Duluth. We ran into rain late in the day and stayed in a motel called Sleepy Hollow about 20 miles short of Superior. It was a very nice motel.
Tuesday in the off-and-on rain we rode through Duluth, Minnesota, and on up to Thunder Bay, Ontario, where we stayed in Thunder Bay KOA. Wednesday we rode to Wawa, Ontario, and stayed the night at the Good Sam campground north of town. Thursday we returned back to the USA and traveled through the Sault again and back to Hoeft State Park to stay the night. Friday we rode the entire east coast of the Lower Peninsula of Michigan including the thumb down to Port Huron and stayed at Algonac State Park. Saturday August 6th we got up and headed for home via U.S. 12 along the southern end of Michigan and back up to the Kalamazoo area.
We had a great time and put a lot of miles on our bikes. We didn't stop at every place we could have due to time restrictions, but we were able to take in a few. Some of the stops were the mountain at Copper Harbor, Grand High Falls, Ouimet Canyon, Agawa pictographs, Agawa Indian Crafts, Soo Locks, Whitefish Point and the Antlers Bar in Sault Ste. Marie, Michigan. We traveled around 2,400 miles total and had very little trouble other than getting stung by a bee near Thunder Bay.
John Rounds, Lawton, Michigan
Frank Roberts, Kalamazoo, Michigan


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text:
Reunited: Black And Brown German Shepherd Dog Female
- Dog ID 149916
- Status Reunited
- Registered 23 Oct 2019
- Name ZOE
- Gender & Breed Female German Shepherd Dog
- Age Young Adult
- Colour Black and brown
- Marks & Scars Her hair is white under her tail
- Tagged Yes
- Microchipped Yes
- Tattooed No
- Date Lost 01 Sep 2019
- Where Lost Cricklewood and hammersmith area
- Lost In Region South East
- Lost In Post Area NW2
- Date Found 21 Nov 2019
- Date Reunited 21 Nov 2019
- Other Info She had a pink collar with little silver bones but it could be removed
- Phone 1 07548346404
- Phone 2 07548346404
- Listed By Patricia
- Views 2956
- Alerts Sent 250
- See more Reunited dogs
- Poster Image
- Extra Image
Sightings and Information
Please post if you have news about this dog. Log in above or register to leave comments or to like them.
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- 2019-11-24 16:23 allimac
- So pleased it is a happy ending. What a sorry tale of complete balls-up from the moment the "friend" took her. Hope you have a fantastic time together. xx
- 2019-11-22 19:54 Sharon - Social Media Team
- Great news x
- 2019-11-22 15:22 Patlucky
- Brilliant news.
- 2019-11-21 22:55 VetsGetScanning
She was handed into a police station. Patricia's friends brother had sold Zoe on for £200.
Since the call on the 1st November Patricia has fought a really hard battle to get her girl back home. Wonderful to see the joy and such a happy family. Thank you everyone who shared.
- 2019-11-15 18:19 Debbie123
- Disgusting how that person can keep your dog when they have only had it 2 minutes is outrageous would they keep your child - keep going u are in the right and Blue X should b ashamed of themselves - it's a shame u didn't get everything changed as soon as she went missing - if a dog is counted as property and u can prove she is yours I don't understand why the law won't help u - they obviously have no moral compass Last edited: 2019-11-15 18:22:08 by Debbie123
- 2019-11-15 17:23 Bo
- I would suggest this is a civil case and the owner needs to take the person they gave the dog to, to civil court. Facts will then be revealed and a judge will decide the outcome.
- 2019-11-15 16:41 Patlucky
- I have written an email to Blue cross.
I got an answer today .
I was not happy with their answer.
- 2019-11-08 18:25 Jenkins-Admin for Found Dogs
- Patricia..please email email@example.com quoting ID 149166 give all info you have and hopefully police coordinator may be able to help.
- 2019-11-08 17:52 Skywalker99
- Doglost police coordinator should be able to help if there is a crn. There's Ian offence called 'theft by finding' .hope you get Zoe back
- 2019-11-08 16:10 Svend
- Take a look at these groups https://www.facebook.com/groups/BringAlfieHome/ and https://www.facebook.com/groups/FindFrankie/ It sounds like you will need to follow a similar course of action, with good legal advice and support.
- 2019-11-08 14:51 VetsGetScanning
Last Friday, 1st November 2019, I got a call from the microchip database PetTrac and the Victoria Blue Cross Hospital. It sounds like the Blue Cross handed my dog Zoe over to someone knowing she was registered as missing on her microchip.
The microchip company said they know where the dog is but they can't give me any information because the dog is not registered to me at my new address and I must update my registration. I called the Blue Cross back and they said they know where my dog is!
I'm in the process of updating my registration to my new address. The microchip is in lockdown so no one else can change it!
What I still don't know to this day is, how they know but say they can't give me information when Zoe is my dog? All they said was they had spoken to the the person who has my dog and he said "I won't give the dog back because I've got too attached to her"! So that's ok then? Zoe is my family dog and I want her home with me!
I have all the papers from the vet I have all the proof that Zoe is my dog.
I didn't give my dog away - I was asking for help from a "friend"!
I am so stressed out heartbroken and desperate to get her back. I don't look at her as property she is my baby girl our family and we really miss her.
Debbie from Vets Get Scanning and SAMPA is trying to help me and I'm turning to social media to raise awareness of the inadequate microchip system which is failing me in every way.
Chiswick and Hammersmith Met Police reference number sent to Karen Harding.
Please sign #FernsLaw petition
Last edited: 2019-11-08 15:31:27 by VetsGetScanning
- 2019-10-24 19:36 GeoffA - Area Volunteer, Cornwall, Scotland & Northumberland
- Photos cropped and framed to squarer image to ensure correct preview shows on shares and to produce a better poster view.
- 2019-10-24 12:44 Unknown
- Confirmed found dog is not Zoe
- 2019-10-24 09:09 sharon57blue
- Is this her ??? https://lostdogsuk.co.uk/wp-content/uploads/2019/10/SDK-ICS1002382.pdf
- 2019-10-23 22:46 Sharon - Social Media Team
- Tweeted @DogLostUK x
- 2019-10-23 22:38 Sharon - Social Media Team
- Shared to our national FB group X
- 2019-10-23 14:12 Jayne - Administrator, Head Office
- Alerts sent to helpers in NW2,3,4,6,9,10,11.


text:
Wednesday, November 12, 2008
A good cup of coffee?
Au Bon Pain is definitely one of Northeastern Students' favorite places. You'll know that by just passing by it at any time during the day: it's always crowded. It might be its great location, it must be the rays of light that come into the local during the day through it's transparent walls. It might be the good service or the tasty pastry and treats you might find there.
I'm not so sure about the coffee though. Not that good for such a great place like Au Bon Pain. I would definitely improve their coffee flavors, not that they don't have enough of them because they actually have many, but its flavors are almost flavorless and taste pretty much the same to me. I could say that is the only thing they could improve (not a small thing though, since they are a coffee place).
Even though I do not think it is the best coffee in town, I would still recommend anyone going there. There is nothing like seating at Au Bon Pain and having a quite breakfast in the morning while watching people pass by.
If you want to find out about other coffee places, check our Caffeinated campus map here
Posted by Belen Bogado at 1:02 PM
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