Control of targeting ligand display by pH-responsive polymers on gold nanoparticles mediates selective entry into cancer cells.

Selective targeting of cells for intracellular delivery of therapeutics represents a major challenge for pharmaceutical intervention in disease. Here we show pH-triggered receptor-mediated endocytosis of nanoparticles via surface ligand exposure. Gold nanoparticles were decorated with two polymers: a 2 kDa PEG with a terminal folate targeting ligand, and a di-block copolymer including a pH-responsive and a hydrophilic block. At the normal serum pH of 7.4, the pH-responsive block (apparent pKa of 7.1) displayed a hydrophilic extended conformation, shielding the PEG-folate ligands, which inhibited cellular uptake of the nanoparticles. Under pH conditions resembling those of the extracellular matrix around solid tumours (pH 6.5), protonation of the pH-responsive polymer triggered a coil-to-globule polymer chain contraction, exposing folate residues on the PEG chains. In line with this, endocytosis of folate-decorated polymer-coated gold nanoparticles in cancer cells overexpressing folate receptor was significantly increased at pH 6.5, compared with pH 7.4. Thus, the tumour acidic environment and high folate receptor expression were effectively exploited to activate cell binding and endocytosis of these nanoparticles. These data provide proof-of-concept for strategies enabling extracellular pH stimuli to selectively enhance cellular uptake of drug delivery vectors and their associated therapeutic cargo.

Underlying sources of cognitive-anatomical variation in multi-modal neuroimaging and cognitive testing.

Healthy adults have robust individual differences in neuroanatomy and cognitive ability not captured by demographics or gross morphology (Luders, Narr, Thompson, & Toga, 2009). We used a hierarchical independent component analysis (hICA) to create novel characterizations of individual differences in our participants (N=190). These components fused data across multiple cognitive tests and neuroanatomical variables. The first level contained four independent, underlying sources of phenotypic variance that predominately modeled broad relationships within types of data (e.g., "white matter," or "subcortical gray matter"), but were not reflective of traditional individual difference measures such as sex, age, or intracranial volume. After accounting for the novel individual difference measures, a second level analysis identified two underlying sources of phenotypic variation. One of these made strong, joint contributions to both the anatomical structures associated with the core fronto-parietal "rich club" network (van den Heuvel & Sporns, 2011), and to cognitive factors. These findings suggest that a hierarchical, data-driven approach is able to identify underlying sources of individual difference that contribute to cognitive-anatomical variation in healthy young adults.

A Phase-Locked Loop Epilepsy Network Emulator.

Most seizure forecasting employs statistical learning techniques that lack a representation of the network interactions that give rise to seizures. We present an epilepsy network emulator (ENE) that uses a network of interconnected phase-locked loops (PLLs) to model synchronous, circuit-level oscillations between electrocorticography (ECoG) electrodes. Using ECoG data from a canine-epilepsy model (Davis et al. 2011) and a physiological entropy measure (approximate entropy or ApEn, Pincus 1995), we demonstrate the entropy of the emulator phases increases dramatically during ictal periods across all ECoG recording sites and across all animals in the sample. Further, this increase precedes the observable voltage spikes that characterize seizure activity in the ECoG data. These results suggest that the ENE is sensitive to phase-domain information in the neural circuits measured by ECoG and that an increase in the entropy of this measure coincides with increasing likelihood of seizure activity. Understanding this unpredictable phase-domain electrical activity present in ECoG recordings may provide a target for seizure detection and feedback control.

Cognitive and anatomical data in a healthy cohort of adults.

We present data from a sample of 190 healthy adults including assessments of 4 cognitive factor scores, 12 cognitive tests, and 115 MRI-assessed neuroanatomical variables (cortical thicknesses, cortical and sub-cortical volumes, fractional anisotropy, and radial diffusivity). These data were used in estimating underlying sources of individual variation via independent component analysis (Watson et al., In press) [25].

Health care delivery: perspectives of young people with chronic illness and their parents.

OBJECTIVE: To identify and compare perceptions of health care service delivery held by young people with chronic illness and their parents. METHODS: A convenience sample of young people with chronic illness and their parents were invited to complete a confidential self-report survey. The adolescents were aged 13-18 years inclusive and attended a specialist medical clinic. RESULTS: Participants comprised 53 young people (response rate 88%, 53/60) and 45 parents (response rate 75%, 45/60). Both young people and their parents rate honesty, confidentiality, having good medical knowledge and good listening skills as the most important qualities for a health care provider. Compared to the parent group, fewer young people report the presence of these desired qualities in their current health care provider. A quarter of young people (25%, 13/53) report they do not always trust health professionals to keep their information confidential, and 19% (10/53) of young people report having withheld information from a health professional due to a lack of trust. Some parents and young people would like to discuss a wider range of health topics, including mental health issues, than they currently do with their health provider. Young people and their parents report limited planning with their current health provider for transition to adult health services. CONCLUSIONS: Young people with chronic illnesses have significant levels of dissatisfaction with the health care they receive; this has the potential to impact on their use of health care services and their health outcomes. There is a need for increased provider awareness of the important qualities of health care service delivery to young people.

Increasing prevalence of type 2 diabetes in adolescents.

OBJECTIVE: The rising prevalence of obesity in childhood and adolescence in North America has been paralleled by the emergence of type 2 diabetes in the adolescent age group. We have examined trends in the prevalence of type 2 diabetes in adolescents attending a diabetes clinic in Auckland, New Zealand. METHODS: Surveys of the prevalence of type 2 diabetes in attendees at the adolescent diabetes clinic at the Auckland Diabetes Centre were undertaken in 1996 and 2002. The proportion of type 2 diabetes in incident cases of diabetes diagnosed between these years was also calculated. RESULTS: The prevalence of type 2 diabetes was 1.8% (2/110) in 1996, and 11.0% (18/163) in 2002 (P = 0.008). Type 2 diabetes accounted for 12.5% (6/48) of incident cases of diabetes in the years 1997-1999, and 35.7% (10/28) of cases in the years 2000-2001, indicating a sharp rise in the incidence (P = 0.017) between the two periods. At diagnosis the mean age of the type 2 diabetes subjects was 15 years and the mean body mass index 34.6 kg/m2. Risk factors for cardiovascular disease were common in the subjects with type 2 diabetes: 85% had dyslipidaemia, 58% had increased albumin excretion rates and 28% had systolic hypertension. CONCLUSIONS: Obesity-related type 2 diabetes now accounts for a substantial proportion of newly recognized diabetes in the adolescent age group - and this proportion is escalating rapidly. Adverse cardiovascular risk factors are prevalent in this population. Public health measures to curtail the rise of obesity in childhood and adolescence are required urgently.

Comparison of health-risk behaviours among students in alternative high schools from New Zealand and the USA.

OBJECTIVES: To describe the prevalence of health-risk behaviours of New Zealand alternative education students, and to compare these behaviours to similar students in the USA. METHODS: Thirty-six alternative education schools in the northern region of New Zealand were surveyed. A total of 269 students completed a youth health questionnaire using laptop computers. These data were compared to data from an equivalent population of alternative education students in the USA. RESULTS: Alternative education students from New Zealand and the USA engage in similar high levels of health-risk behaviours. Female students in New Zealand are at particularly high risk of poor health and social outcomes due to high levels of alcohol and marijuana use, driving under the influence of alcohol and high prevalence of risky sexual behaviours. Such health-risk behaviours place alternative education students at greater risk of some of the leading causes of morbidity and mortality in both youth and adult populations. CONCLUSIONS: Findings from the present study support the need for specific health policies and programmes for alternative high school students. Providers of New Zealand alternative education should be aware that female students are at particularly high risk of many health-risk behaviours.

Adolescents' perceptions of a health survey using multimedia computer-assisted self-administered interview.

OBJECTIVE: To ascertain young people's perceptions of an adolescent health survey when administered by multimedia computer assisted self-administered Interview (M-CASI) through analysis of (1) questionnaire item responses and (2) focus group interviews. SETTING: Auckland, New Zealand, 1999. STUDY TYPE: Pilot testing of a 488-item branching questionnaire delivered using a youth-oriented and user-friendly M-CASI interface in a variety of settings using both desktop and laptop computers. Post pilot focus groups of participants identifying their perceptions and experiences of the survey. SAMPLE: 110 school students aged 12 to 18 years. RESULTS: The mean number of questions answered by participants was 316 with the median time to completion being 48 minutes. On average 65% of the total number of questions were seen and of these 1.5% were deliberately not answered. A high level of acceptability and enjoyment of M-CASI was found in the analysis of focus group responses and agreed with the item responses relating to M-CASI within the questionnaire itself. Participants identified privacy and confidentiality as being particularly important for the honesty of their responses. The passive matrix screens of the computers were popular as they could only be viewed from in front. CONCLUSIONS: M-CASI is an acceptable instrument for the administration of a youth health survey. Laptop computers with passive matrix screens are able to enhance perceptions of privacy and confidentiality, which may improve honesty of responses. IMPLICATIONS: M-CASI is now feasible and offers advantages in health surveying.

Modeling the effects of proteins on pH in plasma.

Stewart's model of plasma acid-base balance (Can. J. Physiol. Pharmacol. 61: 1444-1461, 1983) has three weaknesses in the treatment of weak acids: 1) the combination of all weak acids into one entity, 2) inappropriate chemistry for the protein combination with H+, and 3) undocumented values for the dissociation parameters. The present study models serum albumin acid-base properties by fixed negative charges and the association of H+ with the imidazole side chain of histidine. This model has three parameters: 1) the net negative fixed charge (21 eq/mol), 2) the number of histidine residues (16/mol), and 3) the association constant for the imidazole side chain (1.77 x 10(-7) eq/l), all determined from published values. The model was compared with that of Figge, Mydosh, and Fencl (J. Lab. Clin. Med. 120: 713-719, 1992) and with the pH data of Figge, Rossing, and Fencl (J. Lab. Clin. Med. 117: 453-467, 1991). The predictions of pH were excellent, comparable to those found by Figge, Mydosh, and Fencl. The model has the advantages that its structure and parameter values are supported by the literature and that the acid-base effects of factors modifying protein can be investigated.

Analysis of the paired-tracer method of determining cell uptake.

The paired-tracer method has been used extensively for determining cell uptake of numerous substances, although the method of calculating uptake has no published theoretical support. We have investigated the effect of capillary permeability of the tracers on v, the uptake rate calculated directly from the ratio of tracer venous concentrations. For a simple mathematical model of plasma-tissue movement of lactate and an analytic expression for v, it has been shown that values of v calculated in the first moments after tracer injection depend almost entirely on the differences in tracer permeability-surface area product (PS). The model indicates v would never give the correct value of cell uptake. It is also shown that PS differences alone can explain the published values for lactate uptake obtained from v in skeletal muscle of the rat and dog.

Validity of the computer science and applications (CSA) activity monitor in children.

PURPOSE: The purpose of this study was to evaluate the validity of the CSA activity monitor as a measure of children's physical activity using energy expenditure (EE) as a criterion measure. METHODS: Thirty subjects aged 10 to 14 performed three 5-min treadmill bouts at 3, 4, and 6 mph, respectively. While on the treadmill, subjects wore CSA (WAM 7164) activity monitors on the right and left hips. VO2 was monitored continuously by an automated system. EE was determined by multiplying the average VO2 by the caloric equivalent of the mean respiratory exchange ratio. RESULTS: Repeated measures ANOVA indicated that both CSA monitors were sensitive to changes in treadmill speed. Mean activity counts from each CSA unit were not significantly different and the intraclass reliability coefficient for the two CSA units across all speeds was 0.87. Activity counts from both CSA units were strongly correlated with EE (r = 0.86 and 0.87, P < 0.001). An EE prediction equation was developed from 20 randomly selected subjects and cross-validated on the remaining 10. The equation predicted mean EE within 0.01 kcal.min-1. The correlation between actual and predicted values was 0.93 (P < 0.01) and the SEE was 0.93 kcal.min-1. CONCLUSION: These data indicate that the CSA monitor is a valid and reliable tool for quantifying treadmill walking and running in children.

Measurement of tissue volume during non-steady state high-intensity muscle contraction.

To investigate the pressures driving water into stimulated muscle, water distribution during and after muscle stimulation was studied in isolated cat muscles perfused by recirculating diluted blood. 51Cr-labeled EDTA (51Cr-EDTA) and Evans blue-labeled albumin were used to determine extracellular volume and plasma volume (PV), respectively. Change in tissue volume was calculated as -PV. Interstitial volume (IFV) was determined from the ratio of interstitial solute (51Cr-EDTA and sodium) mass and interstitial concentration. Interstitial mass was determined by mass balance, and interstitial concentration was determined from solute flux and Fick's Law. One group was stimulated at 4 Hz for 2 min, and a second was stimulated by 80-Hz trains (1 train/s, 0.1 s duration). Four Hertz stimulation increased total tissue volume by approximately 3 ml/100 g and decreased IFV by 1 ml/100 g. Train stimulation increased total tissue volume by 6 ml/100 g and decreased IFV by 4. These data indicate that water moves into cells faster than the simultaneous transcapillary flow, suggesting that intracellular osmoles provide the primary driving pressure in stimulation-induced swelling.

Permeability of cat skeletal muscle capillaries to small solutes.

Permeability-surface area products (PS) for 51Cr-EDTA, [3H]mannitol, [14C]urea, and 22Na were measured in isolated, perfused, lower hindlimb muscles of anesthetized cats. The tracers were added stepwise to the arterial inflow, and Evans blue-labeled albumin was the reference indicator. At flow rates > 70 ml.min-1.100 g-1, the PS values (+/- SE) were 5.0 +/- 0.5, 7.6 +/- 1.2, 17.8 +/- 1.4, and 21.0 +/- 1.4 (n = 7, 4, 5, and 3 animals, respectively). The ratio of simultaneous PS measurements of mannitol and urea was 0.42 +/- 0.02 (n = 9), significantly less than the free diffusion coefficient ratio (0.49), indicating the presence of restricted diffusion. PS measurements were also made during osmotic flow (4.2 +/- 0.6 ml.min-1.100 g-1) induced by 20% NaCl. The data clearly showed that osmotic transients did not alter small solute permeability. Pore models were used to show that the PS data and previously reported reflection coefficient data were consistent with a single description of the capillary wall. This model contained a water-only pathway containing 60% of the hydraulic capacity and an extracellular route modeled by pores of 4 nm radius having 21,000 cm of area per unit membrane thickness (A/delta x).

Sieving of electrolytes at capillary wall of cat skeletal muscle by osmotic water flow.

To test the hypothesis that a significant proportion of transcapillary water flow occurs through solute-restricting channels, we investigated the effects of transcapillary water movement on plasma electrolytes in isolated perfused cat skeletal muscle. The lower hindlimbs of anesthetized cats were perfused with a plasma-albumin solution and were weighed to determine transcapillary water movement. Osmolality was increased 60-70 mosmol/kgH2O with sucrose, creating water fluxes of 8-10 ml.min-1.100 g-1, and the changes in the venous concentrations of sodium, potassium, and chloride were determined. The ion concentrations were all reduced by 6-7% with no significant difference between them. The amount of reduction was quantitatively explained by the flow of ion-free water from the interstitial space into plasma and the diffusion of electrolyte in the same direction. These findings support the hypothesis that important water-only transcapillary channels exist in mammalian skeletal muscle. The observations may also explain some of the electrolyte changes seen in intense exercise.

Effect of plasma osmolality on steady-state fluid shifts in perfused cat skeletal muscle.

Fluid redistribution in isolated perfused cat calf muscle caused by rapid increases in plasma osmolality was studied using NaCl or sucrose. Extracellular tracers (51Cr-labeled EDTA or [3H]mannitol) were added to the perfusate 90 min before solutes were added, and samples were taken from plasma immediately before osmolality was increased and 17, 40, and 65 min later. Interstitial fluid volume (IFV) was calculated as extracellular volume (ECV) minus plasma volume (Evans blue dye). Total tissue water changes (delta TTW) were measured by continuous recording of tissue weight. Change in intracellular volume (delta ICV) was obtained from delta TTW--delta IFV. TTW, IFV, ICV, and plasma osmolality were in steady state after 17 min. Changes in hydrostatic and colloid osmotic pressure were insignificant in comparison with small-molecule osmotic pressure changes. The apparent volume of TTW participating in the fluid shift averaged 65 +/- 1 ml/100 g (SE) over a wide range of osmolality increases. In contrast to the large changes in TTW, IFV was not altered by osmolality. Thus decreases in TTW were similar to cell dehydration. Hence, increases in plasma volume induced by hypertonic fluids may come entirely at the expense of cell volume, not interstitial volume.

Weekly variability in total body water using 2H2O dilution in college-age males.

The variability in measuring total body water (TBW) using deuterium oxide (2H2O) dilution has not been extensively tested. The purpose of this study was to determine the variability of weekly measurements of TBW utilizing the 2H2O dilution technique in 10 males and to determine the amount of biological versus technical variability using a generalizability ANOVA (GENOVA). Ten male subjects aged 21-32 yr were tested on four separate occasions for body weight (BW), underwater weight (UwWt), and TBW. BW remained quite stable across the four sessions with a 0.7% coefficient of variation (CV). Both UwWt and TBW had a CV of 4% across the four sessions. No differences were found between TBW estimates from plasma, saliva, or urine. GENOVA analysis determined that 25-50% of the total variability in measuring TBW was attributed to the sample analysis/separation procedures. Four percent of the TBW in these subjects was 1.8 l. This technique would therefore not be expected to detect changes less than 0.9 l (50% of 1.8 l). The estimates of %FAT from TBW were lower but not significantly different from UwWt. Blood, urine, and saliva were able to produce similarly repeatable measures of TBW.

Water uptake in stimulated cat skeletal muscle.

Isolated vasodilated cat hindlimb skeletal muscles were perfused at constant flow and stimulated at 4 Hz for 2-4 min in three studies. Water uptake rates were measured gravimetrically or calculated from venous protein concentration changes. Venous plasma sodium, potassium, chloride, and osmolality were also measured. Maximum water uptake rates averaged 1.8 +/- 0.2 (SE) ml.min-1 x 100 g-1, reaching twice that in some experiments. Water uptake continued after stimulation had ceased. Constant-flow perfusion maintained a constant capillary pressure that was corroborated by measurements of arterial and venous perfusate pressures. Water uptake rate was not influenced by hematocrit but was highly correlated with plasma flow rate. The evidence strongly suggests that small-molecule osmotic pressure was the primary pressure causing the transcapillary water flux. Venous plasma sodium and chloride concentrations increased almost as much as protein (108 and 87% of the protein increase, respectively), as would be expected when water fluxes are driven by small-molecule osmotic pressure. Peak potassium efflux averaged 36 +/- 3 mu eq.min-1 x 100 g-1, but potassium did not contribute significantly to the osmotic gradient.

Transport parameter estimation from lymph measurements and the Patlak equation.

Two methods of estimating protein transport parameters for plasma-to-lymph transport data are presented. Both use IBM-compatible computers to obtain least-squares parameters for the solvent drag reflection coefficient and the permeability-surface area product using the Patlak equation. A matrix search approach is described, and the speed and convenience of this are compared with a commercially available gradient method. The results from both of these methods were different from those of a method reported by Reed, Townsley, and Taylor [Am. J. Physiol. 257 (Heart Circ. Physiol. 26): H1037-H1041, 1989]. It is shown that the Reed et al. method contains a systematic error. It is also shown that diffusion always plays an important role for transmembrane transport at the exit end of a membrane channel under all conditions of lymph flow rate and that the statement that diffusion becomes zero at high lymph flow rate depends on a mathematical definition of diffusion.

Microvascular permeability transients due to histamine in cat limb.

We measured the protein solvent drag reflection coefficient (sigma f) and the capillary filtration coefficient (CFC) before and after adding 1 or 10 microM histamine to the recirculating fluid (20% plasma, remainder albumin and electrolytes, hematocrit of 1-2%) perfusing the isolated cat hindlimb preparation. Transient sigma f measurements were made at 3- to 15-min intervals after histamine using a modification of the steady-state integral-mass balance method. CFC measurements were made at approximately 10-min intervals after histamine in separate experiments. A 1 microM dose of histamine caused sigma f to fall from approximately 0.8 to approximately 0.3 in 2-3 min; sigma f then returned to control in approximately 20 min. CFC response to the 1 microM histamine was a peak increase approximately 2 times control and a return to control in approximately 40 min. A 10 microM dose caused sigma f to fall rapidly to near zero. In general, recovery was much slower than for the 1 microM dose, most of the limbs not returning to control by 40 min after histamine. CFC measurements after 10 microM histamine increased only approximately 5 times control even though sigma f was near zero at the same time. CFC remained above control for approximately 60 min. The combined sigma f and CFC data could be described quantitatively if histamine simultaneously opened both short-lived large gaps (approximately 1,000 A) and a longer-lived pathway that sieved protein like the normal pathway and if the numbers of channels of each pathway closed exponentially with 4- and 15-min time constants, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of elevated venous pressure on capillary permeability in cat hindlimbs.

We investigated the effects of elevated venous pressure, Pv, (up to 140 mmHg) on the solvent drag reflection coefficient, sigma f, for protein and on the capillary filtration coefficient, CFC, in the isolated cat hindlimb perfused at constant flow. The perfusate contained 30% cat plasma and the remainder was a dialyzed albumin-electrolyte mixture. Cat red cells were added to a hematocrit of approximately 2%. sigma f was measured from the changes in hematocrit and plasma protein concentration (Integral-Mass Balance method) resulting from the fluid filtration caused by the Pv elevation. CFC was measured from the slope of the limb weight recording 2-4 min after the Pv elevation. sigma f decreased linearly from 0.807 (Pv less than 50 mmHg) to approximately 0.2 at 140 mmHg. CFC increased linearly from 0.0086 ml.min-1.mmHg-1.100 g-1 to about 0.04 over the same pressure range. A weight-independent filtration coefficient calculated from the change in hematocrit and a measurement of the initial perfusate volume gave comparable results, except at the very highest of pressures, where this coefficient was sometimes 20-40% less than CFC. Successive sigma f determinations at Pv at about 40 mmHg did not return to control after an initial measurement in which Pv was approximately 110 mmHg. Pore-theory analysis of the data suggests that the elevated Pv causes large pores to open as opposed to the stretching of small pores. Also, these large pores may remain open for a period of hours.