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1.  In situ rheology of Staphylococcus epidermidis bacterial biofilms 
Soft matter  2012;9(1):122-131.
We developed a method to grow Staphylococcus epidermidis bacterial biofilms and characterize their rheological properties in situ in a continuously fed bioreactor incorporated into a parallel plate rheometer. The temperature and shear rates of growth modeled bloodstream conditions, a common site of S. epidermidis infection. We measured the linear elastic (G′) and viscous moduli (G″) of the material using small-amplitude oscillatory rheology and the yield stress using non-linear creep rheology. We found that the elastic and viscous moduli of the S. epidermidis biofilm were 11 ± 3 Pa and 1.9 ± 0.5 Pa at a frequency of 1 Hz (6.283 rad per s) and that the yield stress was approximately 20 Pa. We modeled the linear creep response of the biofilm using a Jeffreys model and found that S. epidermidis has a characteristic relaxation time of approximately 750 seconds and a linear creep viscosity of 3000 Pa s. The effects on the linear viscoelastic moduli of environmental stressors, such as NaCl concentration and extremes of temperature, were also studied. We found a non-monotonic relationship between moduli and NaCl concentrations, with the stiffest material properties found at human physiological concentrations (135 mM). Temperature dependent rheology showed hysteresis in the moduli when heated and cooled between 5 °C and 60 °C. Through these experiments, we demonstrated that biofilms are rheologically complex materials that can be characterized by a combination of low modulus (~10 Pa), long relaxation time (~103 seconds), and a finite yield stress (20 Pa). This suggests that biofilms should be viewed as soft viscoelastic solids whose properties are determined in part by local environmental conditions. The in situ growth method introduced here can be adapted to a wide range of biofilm systems and applied over a broad spectrum of rheological and environmental conditions because the technique minimizes the risk of irreversible, non-linear deformation of the microbial specimen before analysis.
PMCID: PMC4276346  PMID: 25544855
2.  Molar mass, entanglement and associations of the biofilm polysaccharide of Staphylococcus epidermidis 
Biomacromolecules  2013;14(5):1474-1481.
Biofilms are microbial communities that are characterized by the presence of a viscoelastic extracellular polymeric substance (EPS). Studies have shown that polysaccharides, along with proteins and DNA, are a major constituent of the EPS, and play a dominant role in mediating its microstructure and rheological properties. Here, we investigate the possibility of entanglements and associative complexes in solutions of extracellular polysaccharide intercellular adhesin (PIA) extracted from Staphylococcus epidermidis biofilms. We report that the weight average molar mass and radius of gyration of PIA isolates are 2.01 × 105 ± 1200 g/mol and 29.2 ± 1.2 nm respectively. The coil overlap concentration, c*, was thus determined to be (32 ± 4) × 10−4 g/mL. Measurements of the in situ concentration of PIA (cPIA,Biofilm) was found to be (10 ± 2) × 10−4 g/mL. Thus, cPIA,Biofilm < c* and the amount of PIA in the biofilm is too low to cause polymer chain entanglements. In the pH range 3.0 to 5.5, PIA was found to both self-associate and to form complexes with bovine serum albumin (BSA). By static light scattering, both self-association and complex formation with 0.5 %(w/v) BSA were found to occur at PIA concentrations of 0.30 × 10−4 g/mL and greater, which is about 30 times lower than the measured cPIA,Biofilm. These results suggest that the microscopic origin of EPS viscoelasticity is unlikely to be due to polysaccharide entanglements. Furthermore, the onset of self-association and protein complexation of PIA occurs at concentrations far lower than the native PIA concentration in biofilms. This finding therefore suggests a critical role for those two association mechanisms in mediating biofilm viscoelasticity.
PMCID: PMC3676870  PMID: 23540609
Staphylococcus epidermidis; biofilm; entangled polymers; polysaccharide-protein complex; extracellular polysaccharides; static light scattering
3.  Preoperative Body Mass Index, 30-Day Postoperative Morbidity, Length of Stay and Quality of Life in Patients Undergoing Pelvic Exenteration Surgery for Recurrent and Locally-Advanced Rectal Cancer 
Annals of Coloproctology  2014;30(2):83-87.
Malnutrition is associated with an increased risk of developing complications following gastrointestinal surgery, especially following radical surgeries such as pelvic exenteration. This study aims to determine if preoperative body mass index (BMI) is associated with 30-day morbidity, length of hospital stay and/or quality of life (QoL) in patients undergoing pelvic exenteration surgery for recurrent and locally-advanced rectal cancer prior to a prospective trial.
A review of all patients who underwent pelvic exenteration surgery prior to 2008 was performed. Patients were included if they had a documented BMI as well as a QoL measurement (Functional Assessment Cancer Therapy - Colorectal questionnaire).
Thirty-one patients, with a mean age of 56 years, had preoperative height and weight data, as well as measures of postoperative QoL, and formed the study group. The numbers of patients with recurrent (n = 17) or locally-advanced rectal cancer (n = 14) were similar. The mean length of stay was 21 days while the mean BMI of the patients was 24.3 (± 5.9) kg/m2. The majority of the patients were either of normal weight (n = 15) or overweight/obese (n = 11). The average length of hospital stay was significantly longer in patients who were underweight compared to those who were of normal weight (F = 6.508, P = 0.006) and those who were overweight and obese (F = 6.508, P = 0.007).
This study suggests that a lower body mass index preoperatively is associated with a longer length of hospital stay. BMI is not associated with long-term QoL in this patient group. However, further prospective research is required.
PMCID: PMC4022757  PMID: 24851218
Body mass index; Rectal neoplasms; Pelvic exenteration; Treatment outcome
4.  Role of Environmental and Antibiotic Stress on Staphylococcus epidermidis Biofilm Microstructure 
Cellular clustering and separation of Staphylococcus epidermidis surface adherent biofilms were found to depend significantly on both antibiotic and environmental stress present during growth under steady flow. Image analysis techniques common to colloidal science were applied to image volumes acquired with high-resolution confocal laser scanning microscopy to extract spatial positions of individual bacteria in volumes of size ~30 × 30 × 15 μm3. The local number density, cluster distribution, and radial distribution function were determined at each condition by analyzing the statistics of the bacterial spatial positions. Environmental stressors of high osmotic pressure (776 mM NaCl) and sublethal antibiotic dose (1.9 μg/mL vancomycin) decreased the average bacterial local number density 10-fold. Device-associated bacterial biofilms are frequently exposed to these environmental and antibiotic stressors while undergoing flow in the bloodstream. Characteristic density phenotypes associated with low, medium, and high local number densities were identified in unstressed S. epidermidis biofilms, while stressed biofilms contained medium- and low-density phenotypes. All biofilms exhibited clustering at length scales commensurate with cell division (~1.0 μm). However, density phenotypes differed in cellular connectivity at the scale of ~6 μm. On this scale, nearly all cells in the high- and medium-density phenotypes were connected into a single cluster with a structure characteristic of a densely packed disordered fluid. However, in the low-density phenotype, the number of clusters was greater, equal to 4% of the total number of cells, and structures were fractal in nature with df =1.7 ± 0.1. The work advances the understanding of biofilm growth, informs the development of predictive models of transport and mechanical properties of biofilms, and provides a method for quantifying the kinetics of bacterial surface colonization as well as biofilm fracture and fragmentation.
PMCID: PMC4144346  PMID: 23688391
5.  Ethanol sclerotherapy of rectal venous abnormalities in Klippel–Trenaunay syndrome 
Journal of Surgical Case Reports  2014;2014(8):rju080.
Klippel–Trenaunay syndrome (KTS) is a rare congenital disorder characterized by the triad of capillary malformations, atypical venous malformations and varicosities and bony and/or soft tissue hypertrophy. We present the case of an 18-year-old man with KTS affected by haematochezia secondary to rectal venous malformations that was managed with endoscopic sclerotherapy. In this case, we compared the use of ethanol to phenol as a sclerosant.
PMCID: PMC4138432  PMID: 25141856
6.  Complement C5a Generation by Staphylococcal Biofilms 
Shock (Augusta, Ga.)  2013;39(4):336-342.
Biofilms production is a central feature of nosocomial infection of catheters and other medical devices used in resuscitation and critical care. However, the very effective biofilm forming pathogen Staphylococcus epidermidis often produces a modest host inflammatory response and few of the signs and symptoms associated with more virulent pathogens. To examine the impact of bacterial biofilm formation on provocation of an innate immune response, we studied the elaboration of the major complement anaphylatoxin C5a by human serum upon contact with S. epidermidis biofilms. Wildtype S. epidermidis and mutants of sarA (a regulatory protein that promotes synthesis of the biofilm-forming polysaccharide intercellular adhesin, PIA) and icaB (responsible for post-export processing of PIA) were studied. C5a release, as a function of exposed biofilm surface area, was on the order of 1 fmol cm−2 sec−1 and was dependent on the presence of PIA. Experimental results were used to inform a physiologically-based pharmacokinetic model of C5a release by an infected central venous catheter, one of S. epidermidis' primary means of causing human disease. These simulations revealed that the magnitude of C5a release on a superior vena cava catheter completely covered with S. epidermidis would be lower than necessary to alert circulating leukocytes. Combined, the experimental and computational results are highly consistent with clinical observations in which the clinical signs of central line associated bloodstream infection are often muted in association with this important pathogen.
PMCID: PMC4096246  PMID: 23459111
7.  Adjuvant chemotherapy for colorectal cancer: age differences in factors influencing patients’ treatment decisions 
Older colorectal cancer patients are significantly less likely than younger patients to receive guideline-recommended adjuvant chemotherapy. Previous research has indicated that patient refusal of treatment is a contributing factor. This study aimed to identify potential barriers to adjuvant chemotherapy use in older patients by examining the associations between patient age, factors influencing chemotherapy treatment decisions, and preferences for information and decision-making involvement.
Patients and methods
Sixty-eight patients who underwent surgery for colorectal cancer in Sydney, Australia, within the previous 24 months completed a self-administered survey.
Fear of dying, health status, age, quality of life, and understanding treatment procedures and effects were significantly more important to older patients (aged ≥65 years) than younger patients in deciding whether to accept chemotherapy (all P < 0.05). Reducing the risk of cancer returning and physician trust were important factors for all patients. Practical barriers such as traveling for treatment and cost were rated lowest. Older patients preferred less information and involvement in treatment decision making than younger patients. However, 60% of the older group wanted detailed information about chemotherapy, and 83% wanted some involvement in decision making. Those preferring less information and involvement still rated many factors as important in their decision making, including understanding treatment procedures and effects.
A range of factors appears to influence patients’ chemotherapy decision making, including, but not limited to, survival benefits and treatment toxicity. For older patients, balancing the risks and benefits of treatment may be made more complex by the impact of emotional motivators, greater health concerns, and conflicts between their need for understanding and their information and decision-making preferences. Through greater understanding of perceived barriers to treatment and unique motivators for treatment choice, physicians may be better able to support older patients to make informed decisions about their care.
PMCID: PMC3755704  PMID: 24003305
preferences; views; decision making; adjuvant therapy; older; elderly
8.  Multicellularity and Antibiotic Resistance in Klebsiella pneumoniae Grown Under Bloodstream-Mimicking Fluid Dynamic Conditions 
The Journal of Infectious Diseases  2012;206(4):588-595.
Background. While the importance of fluid dynamical conditions is well recognized in the growth of biofilms, their role during bacteremia is unknown. We examined the impact of physiological fluid shear forces on the development of multicellular aggregates of Klebsiella pneumoniae.
Methods. Wild-type and O-antigen or capsular mutants of K. pneumoniae were grown as broth culture in a Taylor-Couette flow cell configured to provide continuous shear forces comparable to those encountered in the human arterial circulation (ie, on the order of 1.0 Pa). The size distribution and antibiotic resistance of aggregates formed in this apparatus were determined, as was their ability to persist in the bloodstream of mice following intravenous injection.
Results. Unlike growth in shaking flasks, bacteria grown in the test apparatus readily formed aggregates, a phenotype largely absent in capsular mutants and to a lesser degree in O-antigen mutants. Aggregates were found to persist in the bloodstream of mice. Importantly, organisms grown under physiological shear were found to have an antibiotic resistance phenotype intermediate between that of fully planktonic and biofilm states.
Conclusions. When grown under intravascular-magnitude fluid dynamic conditions, K. pneumoniae spontaneously develops into multicellular aggregates that are capable of persisting in the circulation and exhibit increased antibiotic resistance.
PMCID: PMC3491734  PMID: 22711903
9.  Contribution of the Klebsiella pneumoniae Capsule to Bacterial Aggregate and Biofilm Microstructures▿ †  
We studied the interaction between capsule production and hydrodynamic growth conditions on the internal and macroscopic structure of biofilms and spontaneously formed aggregates of Klebsiella pneumoniae. Wild-type and capsule-deficient strains were studied as biofilms and under strong and mild hydrodynamic conditions. Internal organization of multicellular structures was determined with a novel image-processing algorithm for feature extraction from high-resolution confocal microscopy. Measures included interbacterial spacing and local angular alignment of individual bacteria. Macroscopic organization was measured via the size distribution of aggregate populations forming under various conditions. Compared with wild-type organisms, unencapsulated mutant organisms formed more organized aggregates with less variability in interbacterial spacing and greater interbacterial angular alignment. Internal aggregate structure was not detectably affected by the severity of hydrodynamic growth conditions. However, hydrodynamic conditions affected both wild-type and mutant aggregate size distributions. Bacteria grown under high-speed shaking conditions (i.e., at Reynolds' numbers beyond the laminar-turbulent transition) formed few multicellular aggregates while clumpy growth was common in bacteria grown under milder conditions. Our results indicate that both capsule and environment contribute to the structure of communities of K. pneumoniae, with capsule exerting influence at an interbacterial length scale and fluid dynamic forces affecting overall particle size.
PMCID: PMC3067277  PMID: 21239544
10.  Measuring cancer care coordination: development and validation of a questionnaire for patients 
BMC Cancer  2011;11:298.
Improving the coordination of cancer care is a priority area for service improvement. However, quality improvement initiatives are hindered by the lack of accurate and reliable measures of this aspect of cancer care. This study was conducted to develop a questionnaire to measures patients' experience of cancer care coordination and to assess the psychometric properties of this instrument.
Questionnaire items were developed on the basis of literature review and qualitative research involving focus groups and interviews with cancer patients, carers and clinicians. The draft instrument was completed 686 patients who had been recently treated for a newly diagnosed cancer, including patients from metropolitan, regional and rural areas of New South Wales, Australia. To assess test-retest reliability, 119 patients completed the questionnaire twice. Unreliable items those with limited variability or high levels of missing data were eliminated. Exploratory factor analysis was conducted to define the underlying factor structure of the remaining items and subscales were constructed. Correlations between these and global measures of the experience of care coordination and the quality of care were assessed.
Of 40 items included in the draft questionnaire, 20 were eliminated due to poor test-retest reliability (n = 4), limited response distributions (n = 8), failure to load onto a factor (n = 7) or detrimental effect on the internal consistency of the scale (n = 1). The remaining 20 items loaded onto two factors named 'Communication' and 'Navigation', which explained 91% of the common variance. Internal consistency was with high for the instrument (Cronbach's alpha 0.88) and each subscale (Cronbach's alpha 0.87 and 0.73 respectively). There was no apparent 'floor' or 'ceiling' effect for the total score or the Communication subscale, but evidence of a ceiling effect for the Navigation subscale with 21% of respondents achieving the highest possible score. There were moderate positive associations between the total score and global measures of care coordination (r = 0.57) and quality of care (r = 0.53).
The instrument developed in this study demonstrated consistency and robust psychometric properties. It may provide a useful tool to measure patients' experience of cancer care coordination in future surveys and intervention studies.
PMCID: PMC3151230  PMID: 21756360
cancer; coordination of cancer care; questionnaire; psychometrics
11.  Fundamentals of magnet-actuated droplet manipulation on an open hydrophobic surface† 
Lab on a chip  2009;9(11):1567-1575.
We systematically investigate droplet movement, coalescence, and splitting on an open hydrophobic surface. These processes are actuated by magnetic beads internalized in an oil-coated aqueous droplet using an external magnet. Results are organized into an ‘operating diagram’ that describes regions of droplet stable motion, breakage, and release from the magnet. The results are explained theoretically with a simple model that balances magnetic, friction, and capillary-induced drag forces and includes the effects of particle type, droplet size, surrounding oil layer, surface tension, and viscosity. Finally, we discuss the implications of the results for the design of magnet-actuated droplet systems for applications such as nucleic acid purification, immunoassay and drug delivery.
PMCID: PMC2932710  PMID: 19458864
12.  Flexible microfluidic device for mechanical property characterization of soft viscoelastic solids such as bacterial biofilms 
We introduce a flexible microfluidic device to characterize the mechanical properties of soft viscoelastic solids such as bacterial biofilms. In the device, stress is imposed on a test specimen by application of a fixed pressure to a thin, flexible poly(dimethyl siloxane) (PDMS) membrane that is in contact with the specimen. The stress is applied by pressurizing a microfabricated air channel located above the test area. The strain resulting from the applied stress is quantified by measuring the membrane deflection with a confocal laser-scanning microscope. The deflection is governed by the viscoelastic properties of the PDMS membrane and of the test specimen. The relative contributions of the membrane and test material to the measured deformation are quantified by comparing a finite element analysis and an independent (control) measurement of the PDMS membrane mechanical properties. The flexible microfluidic rheometer was used to characterize both the steady-state elastic modulus and transient strain recoil of two soft materials: gellan gums and bacterial biofilms. The measured linear elastic moduli and viscoelastic relaxation times of gellan gum solutions were in good agreement with the results of conventional mechanical rheometry. The linear Young’s moduli of biofilms of Staphylococcus epidermidis and Klebsiella pneumoniae, which could not be measured using conventional methods, were found to be 3.2 kPa and 1.1 kPa, respectively, and the relaxation time of the S. epidermidis biofilm was 13.8 s. Additionally, strain hardening was observed in all the biofilms studied. Finally, design parameters and detection limits of the method show that the device is capable of characterizing soft viscoelastic solids with elastic moduli in the range of 102 – 105 Pa. The flexible microfluidic rheometer addresses a need for mechanical property characterization of soft viscoelastic solids common in fields such as biomaterials, food and consumer products. It requires only ~ 200 pL of test specimen.
PMCID: PMC2723186  PMID: 19219968
13.  What are the current barriers to effective cancer care coordination? A qualitative study 
National cancer policies identify the improvement of care coordination as a priority to improve the delivery of health services for people with cancer. Identification of the current barriers to effective cancer care coordination is needed to drive service improvement.
A qualitative study was undertaken in which semi-structured individual interviews and focus groups were conducted with those best placed to identify issues; patients who had been treated for a range of cancers and their carers as well as health professionals involved in providing cancer care. Data collection continued until saturation of concepts was reached. A grounded theory influenced approach was used to explore the participants' experiences and views of cancer care coordination.
Overall, 20 patients, four carers and 29 health professionals participated. Barriers to cancer care coordination related to six aspects of care namely, recognising health professional roles and responsibilities, implementing comprehensive multidisciplinary team meetings, transitioning of care: falling through the cracks, inadequate communication between specialist and primary care, inequitable access to health services and managing scarce resources.
This study has identified a number of barriers to coordination of cancer care. Development and evaluation of interventions based on these findings is now required.
PMCID: PMC2891740  PMID: 20482884
15.  Adsorption and elution characteristics of nucleic acids on silica surfaces and their use in designing a miniaturized purification unit 
Analytical biochemistry  2007;373(2):253-262.
We report nucleic acid (NA) adsorption isotherms and elution profiles for silica surfaces and use these to design a miniaturized NA purification unit based on solid-phase extraction with silica beads. The procedure is based on a pressure drop equation for flow through a packed bed and allows estimation of key design parameters such as channel dimensions, liquid flow rates, sample volume and amount of silica needed. The usefulness of this design procedure is shown by applying it to a column-based NA purification device for influenza detection for a case study of Madin-Darby canine kidney (MDCK) cells infected with influenza A virus.
PMCID: PMC2245989  PMID: 18022378
DNA purification; RNA purification; nucleic acid separation; silica; influenza detection; solid-phase-extraction

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