Lack of appreciation of the role of osmotically unresponsive water in cell volume regulation.

The osmotic responsiveness of cell water has been re-evaluated of reports on the osmotic behaviour of cells. In seven animal cell types, the osmotically unresponsive water (OUR) fraction values ranged from 0.75 to 2.41 g water/g dry mass (g/g), and from 25 to 92% of the total cell water. Protein confirmation, aggregation and crowding play a major, but under-recognised, role in determining the extent of OUR and the regulation of cell volume. Volume regulation studies that do not take into account the role of OUR must be judged incomplete.

Maintenance of low sodium and high potassium levels in cells and in tendon/collagen.

Mammalian cells have a higher concentration of potassium and a lower concentration of sodium than their extracellular environment. The mechanisms responsible for the unequal distribution of these ions are commonly ascribed to the presence of an energy requiring plasma membrane ATPase pump, and the presence of membrane channels that pass one ion selectively, while excluding others. This report deals with other mechanisms that might explain this heterogeneous distribution of ions. To study other mechanisms, we turned to a non-living system, specifically tendon/collagen to eliminate the contribution of the membrane pump and channels. A simple gravimetric method was designed to measure solute accumulation or exclusion during rehydration of a well-washed, carefully dried and well-characterized protein specimen (tendon/collagen). Exposure to physiological salt concentrations resulted in selective exclusion of Na+ over K+, whereas exposure to low-salt concentration resulted in accumulation of these solutes. It is postulated that this solute redistribution occurs in all hydrated proteins and is partially responsible for the heterogeneous solute distribution in cells presently assigned to pump and channel mechanisms. Physical and thermodynamic mechanisms are offered to explain the observed heterogeneous solute distributions.

3T magnetic resonance imaging accurately depicts radiofrequency ablation zones in a blood-perfused bovine liver model.

PURPOSE: To determine if noncontrast T1-weighted (T1W) images from 3T magnetic resonance (MR) imaging accurately depict radiofrequency (RF) ablation zones as determined macroscopically and microscopically in a blood-perfused bovine liver model. MATERIALS AND METHODS: Three-dimensional (3D) gradient-recalled echo (GRE) T1W images were obtained on a 3T MR imaging scanner after RF ablations (n = 14) of in vitro blood-perfused bovine livers. The resulting central hypointense and peripheral hyperintense signal regions were measured and compared with the inner tan and outer red zones of the gross specimen. Corresponding ablated hepatic tissue samples were examined microscopically and stained with nicotinamide adenine dinucleotide phosphate (NADPH) to assess for the presence or absence of NADPH diaphorase activity. Bootstrap two-sample hypothesis tests were used to compare MR imaging, gross, and histopathologic measurements. RESULTS: The MR imaging inner ablation zone had a mean radius of 0.80 cm (range 0.33-1.14 cm); the inner zone plus the outer ablation zone had a mean radius of 1.40 cm (range 1.01-1.74 cm). Comparison of the measurements of the inner ablation zone on MR imaging versus the gross specimen showed equivalence (95% confidence interval [CI] -0.122 cm, 0.223 cm). Comparison of the measurements of the outer ablation zone on MR imaging versus the gross and histologic specimens also showed equivalence (95% CI -0.095 cm, 0.244 cm, and -0.146 cm, 0.142 cm). CONCLUSIONS: Noncontrast 3D GRE T1W 3T MR imaging accurately depicts the RF ablation zones in a blood-perfused bovine liver model and can be used as a noninvasive means to assess the 3D morphologic characteristics of RF ablation lesions in the model.

Micro-CT dilatometry measures of molecular collagen hydration using bovine extensor tendon.

PURPOSE: This article introduces a new method to study macromolecular hydration using micro-CT dilatometry. The complexity of hydration dependence on solvent temperature, pH, ionic charge, ionic activity, and ionic radii are barriers to comprehensive understanding of protein function. The crystalline character of collagen-tendon suggests that tendon dilatometry may give direct access to measures of molecular tropocollagen solvation response. METHODS: The molecular basis of the stoichiometric hydration model (SHM) provides tools to validate bovine tendon as a model to study protein-solvent shape response by micro-CT measures of tendon diameter, length, and mass during dehydration. The SHM relates macroscopic properties to molecular properties of water interacting with the surface of collagen molecules. There are marked changes at critical SHM hydration levels h = 0.0653, 0.262, and 0.724 g water/g dry weight. RESULTS: Micro-CT analysis of the length, diameter, and volume combined with gravimetric measures of tendon mass as a function of hydration h (g water/g dry solid) shows asymmetric changes in length, diameter, and density as predicted by SHM. The collagen molecules perturb water properties of polar hydration N=11 waters per tripeptide unit or h approximately 0.724 g/g to confirm MDS prediction of elevated hydration density 20%-50% higher than bulk water. CONCLUSIONS: Results validate the use of tendon dilatometry amplification factors of 10(6)-10(8) as an effective model to investigate protein molecule shape change response to solvent molecules. The tendon model for the first time allows direct study of protein hydration and functional response under physiological conditions.

The molecular stoichiometric hydration model (SHM) as applied to tendon/collagen, globular proteins and cells.

This report describes and documents the presence of multiple water-of-hydration fractions on proteins and in cells. Initial studies of hydration fractions in g of water/g of DM (dry mass) for tendon/collagen led to the development of the molecular SHM (stoichiometric hydration model) and the development of methods for calculating the size of hydration fractions on a number of different proteins of known amino acid composition. The water fractions have differences in molecular motion and other physical properties due to electrostatic interactions of polar water molecules with electric fields generated by covalently bound pairs of opposite partial charge on the protein backbone. The methods allow calculation of the size of four hydration fractions: single water bridges, double water bridges, dielectric water clusters over polar-hydrophilic surfaces and water clusters over hydrophobic surfaces. These four fractions provide monolayer water coverage. The predicted SHM hydration fractions match closely measured hydration fraction values for collagen and for globular proteins. This report also presents water sorption findings that support the SHM. The SHM is applicable for cell systems where it has been studied. In seven cell systems studied, more than half of all of the cell water had properties unlike those of bulk water. The SHM predicts and explains the commonly cited and measured bound water fraction of 0.2-0.4 g of water/g of DM on proteins. The commonly accepted concept that water beyond this bound water fraction can be considered bulk-like water in its physical properties is unwarranted.

QA procedures for multimodality preclinical tumor drug response testing.

PURPOSE: There are growing expectations that imaging biomarkers for tumor therapeutic drug response assessment will speed up preclinical testing of anticancer drugs in rodent models. The only imaging biomarker presently approved by the U.S. Food and Drug Administration is tumor size measurement based on either World Health Organization (WHO) criteria or Response Evaluation Criteria in Solid Tumors (RECIST). Frequently, preclinical data are accumulated from multiple research centers on multiple continents using scanners from different manufacturers and sometimes even using different imaging modalities. Very expensive cancer drug response studies can be compromised by inadequate controls to assure precision and accuracy of tumor size measurements. This project was undertaken to develop standardized quality assurance (QA) procedures using a multimodality preclinical tumor response phantom to validate the accuracy of tumor size measurements based on WHO criteria, RECIST, or global tumor volume criteria for evaluation of cytostatic drugs. METHODS: A tumor response phantom containing five low contrast test objects designed to simulate animal tumor models was made of tissue-mimicking materials. Imaging of the phantom was performed using three modalities in two institutions to evaluate size measurement of tumor-simulating test objects. RESULTS: Evaluation of tumor measurements from the three commonly used imaging devices in two different institutions for monitoring tumor size changes showed that a single phantom for multiple modalities was feasible. The tumor response phantom validated precision and accuracy of tumor response data input from ultrasound, computed tomography, and/or magnetic resonance imaging devices. CONCLUSIONS: Measurement results show that the standardized QA procedures using the tumor response phantom can provide a rationale check of data that excludes input from poorly maintained instruments, inadequate measurement protocols, or random operator error that frequently introduce unacceptable variability or systematic error in multiple institutions trials.

Structure and Dynamics of Collagen Hydration Water from Molecular Dynamics Simulations: Implications of Temperature and Pressure.

Dynamics of water molecules in hydrated collagen plays an important role in determining the structural and functional properties of collagenous tissues. Experimental results suggest that collagen-bridging water molecules exhibit dynamic and thermodynamic properties of one-dimensional ice. However, molecular dynamics (MD) studies performed to date have failed to identify icelike water bridges. It has been hypothesized that this discrepancy is due to the experimental measurements and computational MD analysis having been performed on very different systems: complete tissues with large-scale collagen fiber assemblies and individual tropocollagen fragments, respectively. In this work, we explore ways of emulating a tissuelike macromolecular environment in MD simulations of hydrated collagen without increasing the size of the system to computationally prohibitive levels. We have investigated the effects of temperature and pressure on the dynamics of a small hydrated tropocollagen fragment. The occupancy and bond energies of interchain hydrogen bonds were relatively insensitive to temperature, suggesting that they play a key role in the stability of the collagen triple helix. The lifetimes of water bridges lengthened with decreasing temperature, but even at 280 K, no bridging water molecules exhibited icelike dynamics. We discuss the implications of these findings for the ability to emulate tissuelike conditions in hydrated collagen.

Bioengineering and Imaging Research Opportunities Workshop V: a summary on Imaging and Characterizing Structure and Function in Native and Engineered Tissues.

The Fifth Bioengineering and Imaging Research Opportunities Workshop (BIROW V) was held on January 18-19, 2008. As with previous BIROW meetings, the purpose of BIROW V was to identify and characterize research and engineering opportunities in biomedical engineering and imaging. The topic of this BIROW meeting was Imaging and Characterizing Structure and Function in Native and Engineered Tissues. Under this topic, four areas were explored in depth:1) Heterogeneous single-cell measurements and their integration into tissue and organism models;2) Functional, molecular, and structural imaging of engineered tissue in vitro and in vivo;3) New technologies for characterizing cells and tissues in situ;4) Imaging for targeted cell, gene, and drug delivery.

Characterization of water of hydration fractions in rabbit skeletal muscle with age and time of post-mortem by centrifugal dehydration force and rehydration methods.

Centrifugal dehydration force (CDF) and rehydration isotherm (RHI) methods were used to measure and characterize hydration fractions in rabbit psoas skeletal muscle. The CDF method assessed fluid flow rate from rabbit muscle and hydration capacity of the fractions. Bulk and multiple non-bulk water fractions were identified. The non-bulk water was divisible into the following fractions: two outer non-bulk fractions, a main chain proteins backbone or double water bridge fraction, and a single water bridge fraction. The total non-bulk water amounts to about 85% of the total water in the muscle. The sizes of the water fractions (in g water/g dry mass) agree with a recently proposed molecular stoichiometric hydration model (SHM) applicable to all proteins in and out of cells (Fullerton GD, Cameron IL. Water compartments in cells. Methods Enzymol, 2007; Cameron IL, Fullerton GD. Interfacial water compartments on tendon/collagen and in cells. In: Pollack GH, Chin WC, editors. Phase transitions in cells. Dordrecht, The Netherlands: Springer, 2008). Age of the rabbit significantly slowed the flow rate of the outer non-bulk water fraction by about 50%. Also, muscle of the older rabbit (26 weeks vs. 12 weeks old) had less bulk water and less outer non-bulk water but the same amount of main chain backbone water compared to muscle of the younger rabbit. Increase in time post-mortem from 30min to 4h resulted in rigor mortis and a significantly slower flow rate of water from the outer non-bulk water fraction, which is attributed to muscle contraction, increased packing of contractile elements and increased obstructions to flow of fluid from the muscle fibers.

Water compartments in cells.

Human experience in the macrobiological world leads scientists to visualize water compartments in cells analogous to the bladder in the human pelvis or ventricles in the brain. While such water-filled cellular compartments likely exist, the volume contributions are insignificant relative to those of biomolecular hydration compartments. The purpose of this chapter is to identify and categorize the molecular water compartments caused by proteins, the primary macromolecular components of cells. The categorical changes in free energy of water molecules on proteins cause these compartments to play dominant roles in osmoregulation and provide important adjuncts to fundamental understanding of osmosensing and osmosignaling mechanisms. Water compartments possess differences in molecular motion, enthalpy, entropy, freezing point depression, and other properties because of electrostatic interaction of polar water molecules with electric fields generated by covalently bound pairs of opposite charge caused by electronegative oxygen and nitrogen atoms of the protein. Macromolecules, including polypeptides, polynucleotides, and polysaccharides, are stiff molecular chains with restricted folding capacities due to inclusion of rigid ring structures or double amide bonds in the backbone sequence. This creates "irreducible spatial charge separation" between positive and negative partial charges, causing elevated electrostatic energy. In the fully hydrated in vivo state of living cells the high dielectric coefficient of water reduces protein electrostatic free energy by providing polar "water bridge networks" between charges, thereby creating four measurably different compartments of bound water with distinct free energy differences.

Biomedical imaging research opportunities workshop IV: a white paper.

The Fourth Biomedical Imaging Research Opportunities Workshop (BIROW IV) was held on February 24-25, 2006, in North Bethesda, MD. The workshop focused on opportunities for research and development in four areas of imaging: imaging of rodent models; imaging in drug development; imaging of chronic metabolic disease: diabetes; and image guided intervention in the fourth dimension-time. These topics were examined by four keynote speakers in plenary sessions and then discussed in breakout sessions devoted to identifying research opportunities and challenges in the individual topics. This paper synthesizes these discussions into a strategy for future research directions in biomedical imaging.

Calculation of effective dose from measurements of secondary neutron spectra and scattered photon dose from dynamic MLC IMRT for 6 MV, 15 MV, and 18 MV beam energies.

Effective doses were calculated from the delivery of 6 MV, 15 MV, and 18 MV conventional and intensity-modulated radiation therapy (IMRT) prostate treatment plans. ICRP-60 tissue weighting factors were used for the calculations. Photon doses were measured in phantom for all beam energies. Neutron spectra were measured for 15 MV and 18 MV and ICRP-74 quality conversion factors used to calculate ambient dose equivalents. The ambient dose equivalents were corrected for each tissue using neutron depth dose data from the literature. The depth corrected neutron doses were then used as a measure of the neutron component of the ICRP protection quantity, organ equivalent dose. IMRT resulted in an increased photon dose to many organs. However, the IMRT treatments resulted in an overall decrease in effective dose compared to conventional radiotherapy. This decrease correlates to the ability of an intensity-modulated field to minimize dose to critical normal structures in close proximity to the treatment volume. In a comparison of the three beam energies used for the IMRT treatments, 6 MV resulted in the lowest effective dose, while 18 MV resulted in the highest effective dose. This is attributed to the large neutron contribution for 18 MV compared to no neutron contribution for 6 MV.

Investigation of secondary neutron dose for 18 MV dynamic MLC IMRT delivery.

Secondary neutron doses from the delivery of 18 MV conventional and intensity modulated radiation therapy (IMRT) treatment plans were compared. IMRT was delivered using dynamic multileaf collimation (MLC). Additional measurements were made with static MLC using a primary collimated field size of 10 x 10 cm2 and MLC field sizes of 0 x 0, 5 x 5, and 10 x 10 cm2. Neutron spectra were measured and effective doses calculated. The IMRT treatment resulted in a higher neutron fluence and higher dose equivalent. These increases were approximately the ratio of the monitor units. The static MLC measurements were compared to Monte Carlo calculations. The actual component dimensions and materials for the Varian Clinac 2100/2300C including the MLC were modeled with MCNPX to compute the neutron fluence due to neutron production in and around the treatment head. There is excellent agreement between the calculated and measured neutron fluence for the collimated field size of 10 x 10 cm2 with the 0 x 0 cm2 MLC field. Most of the neutrons at the detector location for this geometry are directly from the accelerator head with a small contribution from room scatter. Future studies are needed to investigate the effect of different beam energies used in IMRT incorporating the effects of scattered photon dose as well as secondary neutron dose.

Measurements of secondary neutron dose from 15 MV and 18 MV IMRT.

Secondary neutron dose-equivalents were determined for conventional and intensity modulated radiation therapy (IMRT) prostate treatments for 15 and 18 MV X-ray beams. Conventional and IMRT treatment plans were generated to deliver 45 Gy to the prostate, seminal vessicles and external and internal iliac lymph nodes. Neutron spectra were determined by unfolding measurements from a TLD-based Bonner sphere system. Treatments using 18 MV IMRT and conventional plans result in neutron ambient dose-equivalents of 687 and 112 mSv, respectively. Delivery of the 15 MV IMRT and conventional plans results in neutron ambient dose-equivalents of 327 and 52 mSv, respectively. The data illustrate that using lower photon energies for IMRT reduces the secondary neutron dose, while still achieving comparable treatment volume coverage and sparing critical normal tissue.

Construction of a preclinical multimodality phantom using tissue-mimicking materials for quality assurance in tumor size measurement.

World Health Organization (WHO) and the Response Evaluation Criteria in Solid Tumors (RECIST) working groups advocated standardized criteria for radiologic assessment of solid tumors in response to anti-tumor drug therapy in the 1980s and 1990s, respectively. WHO criteria measure solid tumors in two-dimensions, whereas RECIST measurements use only one-dimension which is considered to be more reproducible (1, 2, 3,4,5). These criteria have been widely used as the only imaging biomarker approved by the United States Food and Drug Administration (FDA) (6). In order to measure tumor response to anti-tumor drugs on images with accuracy, therefore, a robust quality assurance (QA) procedures and corresponding QA phantom are needed. To address this need, the authors constructed a preclinical multimodality (for ultrasound (US), computed tomography (CT) and magnetic resonance imaging (MRI)) phantom using tissue-mimicking (TM) materials based on the limited number of target lesions required by RECIST by revising a Gammex US commercial phantom (7). The Appendix in Lee et al. demonstrates the procedures of phantom fabrication (7). In this article, all protocols are introduced in a step-by-step fashion beginning with procedures for preparing the silicone molds for casting tumor-simulating test objects in the phantom, followed by preparation of TM materials for multimodality imaging, and finally construction of the preclinical multimodality QA phantom. The primary purpose of this paper is to provide the protocols to allow anyone interested in independently constructing a phantom for their own projects. QA procedures for tumor size measurement, and RECIST, WHO and volume measurement results of test objects made at multiple institutions using this QA phantom are shown in detail in Lee et al. (8).

Ultrasound-guided intratumoral administration of collagenase-2 improved liposome drug accumulation in solid tumor xenografts.

PURPOSE: To investigate the effect of intratumoral administration of collagenase-2 on liposomal drug accumulation and diffusion in solid tumor xenografts. METHODS: Correlation between tumor interstitial fluid pressure (IFP) and tumor physiological properties (size and vessel fraction by B-mode and Doppler ultrasound, respectively) was determined. IFP response to intravenous or intratumoral collagenase-2 (0.1%) treatment was compared with intratumoral deactivated collagenase-2. To evaluate drug accumulation and diffusion, technetium-99 m-((99m)Tc)-liposomal doxorubicin (Doxil) was intravenously injected after collagenase-2 (0.1 and 0.5%, respectively) treatment, and planar scintigraphic images acquired and percentage of the injected dose per gram tissue calculated. Subsequently, tumors were subjected to autoradiography and histopathology. RESULTS: IFP in two-week-old head and neck squamous cell carcinoma xenografts was 18 ± 3.7 mmHg and not correlated to the tumor size but had reverse correlation with the vessel fraction (r = -0.91, P < 0.01). Intravenous and intratumoral collagenase-2 use reduced IFP by a maximum of 35-40%. Compared to the control, the low IFP level achieved through intratumoral route remained for a long period (24 vs. 2 h, P < 0.05). SPECT images and autoradiography showed significantly higher (99m)Tc-Doxil accumulation in tumors with intratumoral collagenase-2 treatment, confirmed by %ID/g in tumors (P < 0.05), and pathological findings showed extensive distribution of Doxil in tumors. CONCLUSIONS: Intratumoral injection of collagenase-2 could effectively reduce IFP in HNSCC xenografts for a longer period than using intravenous approach, which allowed for more efficient accumulation and homogeneous diffusion of the Doxil within the tumor interstitium.

Preclinical multimodality phantom design for quality assurance of tumor size measurement.

BACKGROUND: Evaluation of changes in tumor size from images acquired by ultrasound (US), computed tomography (CT) or magnetic resonance imaging (MRI) is a common measure of cancer chemotherapy efficacy. Tumor size measurement based on either the World Health Organization (WHO) criteria or the Response Evaluation Criteria in Solid Tumors (RECIST) is the only imaging biomarker for anti-cancer drug testing presently approved by the United States Food and Drug Administration (FDA). The aim of this paper was to design and test a quality assurance phantom with the capability of monitoring tumor size changes with multiple preclinical imaging scanners (US, CT and MRI) in order to facilitate preclinical anti-cancer drug testing. METHODS: Three phantoms (Gammex/UTHSCSA Mark 1, Gammex/UTHSCSA Mark 2 and UTHSCSA multimodality tumor measurement phantom) containing tumor-simulating test objects were designed and constructed. All three phantoms were scanned in US, CT and MRI devices. The size of test objects in the phantoms was measured from the US, CT and MRI images. RECIST, WHO and volume analyses were performed. RESULTS: The smaller phantom size, simplified design and better test object CT contrast of the UTHSCSA multimodality tumor measurement phantom allowed scanning of the phantom in preclinical US, CT and MRI scanners compared with only limited preclinical scanning capability of Mark 1 and Mark 2 phantoms. For all imaging modalities, RECIST and WHO errors were reduced for UTHSCSA multimodality tumor measurement phantom (≤1.69 ± 0.33%) compared with both Mark 1 (≤ -7.56 ± 6.52%) and Mark 2 (≤ 5.66 ± 1.41%) phantoms. For the UTHSCSA multimodality tumor measurement phantom, measured tumor volumes were highly correlated with NIST traceable design volumes for US (R2 = 1.000, p < 0.0001), CT (R2 = 0.9999, p < 0.0001) and MRI (R2 = 0.9998, p < 0.0001). CONCLUSIONS: The UTHSCSA multimodality tumor measurement phantom described in this study can potentially be a useful quality assurance tool for verifying radiologic assessment of tumor size change during preclinical anti-cancer therapy testing with multiple imaging modalities.