Peptides@mica: from affinity to adhesion mechanism.

Investigating the adsorption of peptides on inorganic surfaces, on the molecular level, is fundamental for medicinal and analytical applications. Peptides can be potent as linkers between surfaces and living cells in biochips or in implantation medicine. Here, we studied the adsorption process of the positively charged pentapeptide RTHRK, a recently identified binding sequence for surface oxidized silicon, and novel analogues thereof to negatively charged mica surfaces. Homogeneous formation of monolayers in the nano- and low micromolar peptide concentration range was observed. We propose an alternative and efficient method to both quantify binding affinity and follow adhesion behavior. This method makes use of the thermodynamic relationship between surface coverage, measured by atomic force microscopy (AFM), and the concomitant free energy of adhesion. A knowledge-based fit to the autocorrelation of the AFM images was used to correct for a biased surface coverage introduced by the finite lateral resolution of the AFM. Binding affinities and mechanisms were further explored by large scale molecular dynamics (MD) simulations. The combination of well validated MD simulations with topological data from AFM revealed a better understanding of peptide adsorption processes on the atomistic scale. We demonstrate that binding affinity is strongly determined by a peptide's ability to form salt bridges and hydrogen bonds with the surface lattice. Consequently, differences in hydrogen bond formation lead to substantial differences in binding affinity despite conservation of the peptide's overall charge. Further, MD simulations give access to relative changes in binding energy of peptide variations in comparison to a lead compound.

CEST-FISP: a novel technique for rapid chemical exchange saturation transfer MRI at 7 T.

Chemical exchange saturation transfer (CEST) and magnetization transfer techniques provide unique and potentially quantitative contrast mechanisms in multiple MRI applications. However, the in vivo implementation of these techniques has been limited by the relatively slow MRI acquisition techniques, especially on high-field MRI scanners. A new, rapid CEST-fast imaging with steady-state free precession technique was developed to provide sensitive CEST contrast in ∼20 sec. In this study at 7 T with in vitro bovine glycogen samples and initial in vivo results in a rat liver, the CEST-fast imaging with steady-state free precession technique was shown to provide equivalent CEST sensitivity in comparison to a conventional CEST-spin echo acquisition with a 50-fold reduction in acquisition time. The sensitivity of the CEST-fast imaging with steady-state free precession technique was also shown to be dependent on k-space encoding with centric k-space encoding providing a 30-40% increase in CEST sensitivity relative to linear encoding for 256 or more k-space lines. Overall, the CEST-fast imaging with steady-state free precession acquisition technique provides a rapid and sensitive imaging platform with the potential to provide quantitative CEST and magnetization transfer imaging data.

Taxonomic differences in the scaling of brain on body weight among mammals.

Theories for the evolution of brain weight in mammals suggest that closely related species have diverged largely as a result of selection for differences in body weight, but that differences among more distantly related species have arisen due to greater net directional selection on brain weight. This pattern of changing selection causes brain weight to evolve more slowly than body weight among closely related species, such as those in the same genus, than among more distantly related species, such as those from different families or orders; a phenomenon known as the "taxon-level effect." Thus, brain weight differs more for a given difference in body weight as the species compared are more distantly related. An alternative explanation for the taxon-level effect is proposed. Distantly related species are more likely to inhabit different ecological conditions than are more closely related species. Where the taxon-level effect occurs, brain weight appears to have evolved in response to the demands of these different ecological conditions. As a consequence, brain weight differs more among distantly related species, for any given difference in body weight, than among closely related species. This effect, rather than a progressive pattern of changing selection pressures, may account for the taxon-level effect in mammals.

The distribution of blood flow, oxygen consumption, and work output among the respiratory muscles during unobstructed hyperventilation.

An animal model was developed to describe respiratory muscle work output, blood flow, and oxygen consumption during mechanical ventilation, resting spontaneous ventilation, and the increased unobstructed ventilatory efforts induced by CO2 rebreathing. Almost all of the work of breathing was inspiratory work at all ventilatory levels; thus, only blood flows to the diaphragm and external intercostals increased in the transition from mechanical to spontaneous ventilation, and they further increased linearly as ventilatory work was incrementally augmented ninefold by CO2 rebreathing. No other muscles of inspiration manifest increased blood flows. A small amount of expiratory work was measured at high ventilatory volumes during which two expiratory muscles (transverse abdominal and intercostals) had moderate increases in blood flow. Blood pressure did not change, but cardiac output doubled. Arterial-venous oxygen content difference across the diaphragm increased progressively, so oxygen delivery was augmented by both increased blood flow and increased oxygen extraction at all work loads. Oxygen consumption increased linearly as work of breathing increased, so efficiency did not change significantly. The mean efficiency of the respiratory muscles was 15.5%. These results differ significantly from the patterns previously observed by us during increased work of breathing induced by inspiratory resistance, suggesting a different distribution of work load among the various muscles of respiration, a different fractionation of oxygen delivery between blood flow and oxygen extraction, and a higher efficiency when shortening, not tension development, of the muscle is increased.

Characterization of a new model of GM2-gangliosidosis (Sandhoff's disease) in Korat cats.

We have detected a disorder in Korat cats (initially imported from Thailand) that is analogous to human Sandhoff's disease. Pedigree analysis indicates that this disease in an autosomal recessive disorder in the American Korat. Postmortem studies on one affected cat showed hepatomegaly that was not reported in the only other known feline model of GM2-gangliosidosis type II. Histologic and ultra-structural evaluation revealed typical storage vacuoles. There was a marked deficiency in the activity of hexosaminidase (HEX) A and B in affected brain and liver as compared to controls. Electrophoresis of a liver extract revealed a deficiency of normal HEX A and B in the affected animals. The blocking primary enzyme immunoassay verified the presence of antigenically reactive HEX present in affected cat livers in quantities slightly elevated with respect to the normal HEX concentration in control cats. In leukocytes, obligate heterozygotes had intermediate levels of total HEX activity with a slight increase in the percent activity due to HEX A. Indeed, 4 of 11 phenotypically normal animals in addition to four obligate heterozygotes appear to be carriers using this assay. Affected brain and liver compared with control brain and liver contained a great excess of bound N-acetylneuraminic acid in the Folch upper-phase solids; thin-layer chromatography showed a marked increase in GM2-ganglioside. In summary, we have characterized the pedigree, pathology, and biochemistry of a new feline model of GM2-gangliosidosis which is similar to but different from the only other known feline model.

Enhancing the cytotoxicity of chemoradiation with radiation-guided delivery of anti-MGMT morpholino oligonucleotides in non-methylated solid tumors.

The DNA repair enzyme O6-methylguanine DNA methyltransferase (MGMT) is epigenetically silenced in some tumors by MGMT gene promoter methylation. MGMT-hypermethylated solid tumors have enhanced susceptibility to the cytotoxic effects of alkylating chemotherapy such as temozolomide, compared with non-methylated tumors. In glioblastoma, subjects with MGMT hypermethylation have significantly longer survival rates after chemoradiotherapy. We report the first successful use of a non-ablative dose of ionizing radiation to prime human cancer cells to enhance the uptake of unmodified anti-MGMT morpholino oligonucleotide (AMON) sequences. We demonstrate >40% reduction in the in vitro proliferation index and cell viability in radiation-primed MGMT-expressing human solid tumor cells treated with a single dose of AMONs and temozolomide. We further demonstrate the feasibility of using a non-ablative dose of radiation in vivo to guide and enhance the delivery of intravenously administered AMONs to achieve 50% MGMT knockdown only at radiation-primed tumor sites in a subcutaneous tumor model. Local upregulation of physiological endocytosis after radiation may have a role in radiation-guided uptake of AMONs. This approach holds direct translational significance in glioblastoma and brain metastases where radiation is part of the standard of care; our approach to silence MGMT could overcome the significant problem of MGMT-mediated chemoresistance.

Pharmacology and toxicity of intracarotid adriamycin administration following osmotic blood-brain barrier modification.

The effect of reversible blood-brain barrier modification on the delivery of Adriamycin to the brain was studied in a rodent and canine model. Pharmacokinetic and physiological studies were done in these animals after a wide range of doses of Adriamycin (0.1 to 1.0 mg/kg) were administered into the carotid artery following osmotic barrier modification with mannitol. In the absence of barrier modification, no immunoreactive Adriamycin was detected in the cerebrum; whereas, following barrier modification, up to 4.5 micrograms of drug and/or metabolites per g of brain were found. Optimum tissue levels of Adriamycin and metabolites were achieved following barrier modification when the drug was administered by either bolus or slow continuous (15-min) infusion. Immunoreactive drug was identified in brain for up to 6 hr after administration. Significant functional neurotoxicity occurred at all dose levels, even at 0.1 mg/kg, a level at which Adriamycin concentration in the brain was below the level of detectability. Neuropathological examination revealed the presence of necrosis and hemorrhagic infarcts. Thus, these pharmacological and toxicity studies suggest that Adriamycin (or its metabolites) may produce significant clinical neurotoxicity when even small amounts penetrate the blood-brain barrier.

Therapeutic efficacy of aortic administration of N-acetylcysteine as a chemoprotectant against bone marrow toxicity after intracarotid administration of alkylators, with or without glutathione depletion in a rat model.

Modulation of thiol levels may alter both the efficacy and toxicity of chemotherapeutic agents. We investigated cytoenhancement, using L-buthionine-[S,R]-sulfoximine (BSO) to reduce cellular glutathione levels prior to intracarotid alkylator administration. We also evaluated chemoprotection against chemotherapy-induced systemic toxicity when the thiol agents N-acetylcysteine (NAC) and sodium thiosulfate were administered into the descending aorta to limit brain delivery. BSO treatment reduced rat brain and intracerebral tumor glutathione levels by 50-65%, equivalent to the reduction in liver and s.c. tumor. BSO treatment significantly enhanced the toxicity of chemotherapy with carboplatin, melphalan, and etoposide phosphate against granulocytes, total white cells, and platelets. Intracarotid administration of NAC resulted in high delivery to the brain, whereas infusion via the descending aorta minimized brain delivery. When NAC, with or without sodium thiosulfate, was administered via aortic infusion prior to chemotherapy, the magnitude of the bone marrow toxicity nadir was minimized, even with BSO-enhanced myelosuppression. Thus, BSO depleted brain and brain tumor glutathione but thereby increased chemotherapy-induced myelosuppression. Surprisingly, although NAC was found to readily cross the blood-brain barrier when given into the carotid artery, aortic infusion of NAC resulted in minimal exposure to the central nervous system (CNS) vasculature because of rapid clearance. As a result, aortic infusion of NAC to perfuse bone marrow and minimize myelosuppression and toxicity to visceral organs could be performed without interfering with the CNS cytotoxicity of intracarotid alkylators, even after BSO depletion of CNS glutathione.

In vitro and animal studies of sodium thiosulfate as a potential chemoprotectant against carboplatin-induced ototoxicity.

When carboplatin (cis-diammine-1,1-cyclobutane-dicarboxylato-platinum) delivery to brain tumors is optimized with osmotic blood-brain barrier disruption (BBBD), high frequency hearing loss can result. Treatment with sodium thiosulfate (STS) blocked carboplatin cytotoxicity against the LX-1 human small cell lung carcinoma cell line in vitro. STS decreased carboplatin-induced ototoxicity in a guinea pig model, as determined by electrophysiological measurements and analysis of inner ear outer hair cell numbers. Protection was found when STS was administered up to 8 h subsequent to carboplatin but not 24 h after carboplatin. In a rat model of osmotic BBBD, STS was neurotoxic when given immediately after BBBD but not when given 60 min after BBBD, when the barrier is reestablished. Thus, delayed administration of STS may provide a mechanism to reduce the cochlear toxicity caused by BBBD-enhanced carboplatin delivery to the brain.

Relationships between protein structure and dynamics from a database of NMR-derived backbone order parameters.

The amplitude of protein backbone NH group motions on a time-scale faster than molecular tumbling may be determined by analysis of (15)N NMR relaxation data according to the Lipari-Szabo model free formalism. An internet-accessible database has been compiled containing 1855 order parameters from 20 independent NMR relaxation studies on proteins whose three-dimensional structures are known. A series of statistical analyses has been performed to identify relationships between the structural features and backbone dynamics of these proteins. Comparison of average order parameters for different amino acid types indicates that amino acids with small side-chains tend to have greater backbone flexibility than those with large side-chains. In addition, the motions of a given NH group are also related to the sizes of the neighboring amino acids in the primary sequence. The secondary structural environment appears to influence backbone dynamics relatively weakly, with only subtle differences between the order parameter distributions of loop structures and regular hydrogen bonded secondary structure elements. However, NH groups near helix termini are more mobile on average than those in the central regions of helices. Tertiary structure influences are also relatively weak but in the expected direction, with more exposed residues being more flexible on average than residues that are relatively inaccessible to solvent.

Delayed administration of sodium thiosulfate in animal models reduces platinum ototoxicity without reduction of antitumor activity.

Platinum-based chemotherapeutic agents, such as carboplatin and cisplatin, are effective against many human tumors, but their use may be limited by a high incidence of ototoxicity. Delayed administration of the chemoprotective agent sodium thiosulfate (STS) reduces the ototoxicity of carboplatin in a guinea-pig model, when given up to 8 h after the chemotherapy, and also reduces hearing loss in patients given carboplatin with osmotic blood-brain barrier opening for treatment of brain tumors. We tested whether STS, given at times that achieved otoprotection, could impact the chemotherapeutic efficacy of carboplatin. The impact of STS was evaluated by measuring the onset of growth of LX-1 human small cell lung carcinoma s.c. xenografts in the nude rat. When STS was administered as two boluses, 2 and 6 h after treatment with carboplatin and etoposide, there was a decrease in the time to tumor progression. In contrast, when STS administration was delayed until 8 h after carboplatin/etoposide, there was no reduction in the antitumor cytotoxicity of the chemotherapy. STS infusion did not significantly affect ultrafilterable platinum pharmacokinetics in the guinea pig. To explore the potential wider applicability of STS, in a pilot study we tested its efficacy against cisplatin ototoxicity. Delayed administration of STS, 2 h after cisplatin, was protective against cisplatin-induced ototoxicity in the guinea pig model, as determined by electrophysiological measures. On the basis of these data, we suggest that delayed administration of STS may provide a mechanism to reduce the ototoxicity caused by administration of carboplatin or cisplatin for both central nervous system and systemic cancer chemotherapy.

The origins of sexual dimorphism in body size in ungulates.

Jarman (1974) proposed a series of relationships between habitat use, food dispersion, and social behavior and hypothesized a series of evolutionary steps leading to sexual dimorphism in body size through sexual selection in African antelope species. The hypothesis states that sexual size dimorphism evolved in a three-step process. Initially, ancestral monomorphic and monogamous ungulate species occupying closed habitats radiated into open grassland habitats. Polygynous mating systems then rapidly evolved in response to the aggregation of males and females, perhaps in relation to the clumped distribution of food resources in open habitats. Subsequently, size dimorphism evolved in those species occupying open habitats, but not in species that remained in closed habitats or retained monogamy. This hypothesis has played an important role in explaining the origins of sexual dimorphism in mammals. However, the temporal sequence of the events that Jarman proposed has never been demonstrated. Here we use a phylogeny of extant ungulate species, along with maximum-likelihood statistical techniques, to provide a test of Jarman's hypothesis.

A small molecule antagonist of the alpha(v)beta3 integrin suppresses MDA-MB-435 skeletal metastasis.

INTRODUCTION: Breast cancer is one of the most common malignancies affecting women in the United States and Europe. Approximately three out of every four women with breast cancer develop metastases in bone which, in turn, diminishes quality of life. The alpha(v)beta3 integrin has previously been implicated in multiple aspects of tumor progression, metastasis and osteoclast bone resorption. Therefore, we hypothesized that the alpha(v)beta3-selective inhibitor, S247, would decrease the development of osteolytic breast cancer metastases. MATERIALS AND METHODS: Cells were treated in vitro with S247 and assessed for viability and adhesion to matrix components. Athymic mice received intracardiac (left ventricle) injections of human MDA-MB-435 breast carcinoma cells expressing enhanced green-fluorescent protein. Mice were treated with vehicle (saline) or S247 (1, 10, or 100 mg/kg/d) using osmotic pumps beginning either one week before or one week after tumor cell inoculation. Bones were removed and examined by fluorescence microscopy and histology. The location and size of metastases were recorded. RESULTS AND CONCLUSIONS: IC50 for S247 adhesion to alpha(v)beta3 or alpha(IIB)beta3a substrates was 0.2 nM vs. 244 nM, respectively. Likewise, S247 was not toxic at doses up to 1000 microM. However, osteoclast cultures treated with S247 exhibited marked morphological changes and impaired formation of the actin sealing zone. When S247 was administered prior to tumor cells, there was a significant, dose-dependent reduction (25-50% of vehicle-only-treated mice; P = 0.002) in osseous metastasis. Mice receiving S247 after tumor cell inoculation also developed fewer bone metastases, but the difference was not statistically significant. These data suggest that, in the MDA-MB-435 model, the alpha(v)beta3 integrin plays an important role in early events (e.g., arrest of tumor cells) in bone metastasis. Furthermore, the data suggest that alpha(v)beta3 inhibitors may be useful in the treatment and/or prevention of breast cancer metastases in bone.

Inferring the origins of state-dependent courtship traits.

We examine a simple model of state-dependent (indicator) traits that focuses on their evolutionary origins as courtship signals. A necessary condition for the initial evolution of signals was found: the marginal female preference for minimal traits must exceed a certain threshold, where that threshold is proportional to the marginal male fitness costs for minimal traits. We interpret a positive threshold as implying a need for preexisting sensory bias in order to overcome the threshold if indicator signals are to start to evolve. We extend the model to allow for the possibility that signal costs and female preferences may vary over evolutionary time. If there is independent information on the way that signaling costs have evolved, then one may use measurements of contemporary female preferences to make inferences concerning the presence of the ancestral threshold. It is the marginal female preferences for minimal male traits that are important, whereas reconstructing ancestral origins from measurement of average size signals is not informative. Our analyses suggest two foci for future studies: measurement of the marginal response of contemporary females to minimal male signals and reconstruction of how signaling costs have changed over evolutionary time.

Phylogenetic analysis and comparative data: a test and review of evidence.

The question is often raised whether it is statistically necessary to control for phylogenetic associations in comparative studies. To investigate this question, we explore the use of a measure of phylogenetic correlation, lambda, introduced by Pagel (1999), that normally varies between 0 (phylogenetic independence) and 1 (species' traits covary in direct proportion to their shared evolutionary history). Simulations show lambda to be a statistically powerful index for measuring whether data exhibit phylogenetic dependence or not and whether it has low rates of Type I error. Moreover, lambda is robust to incomplete phylogenetic information, which demonstrates that even partial information on phylogeny will improve the accuracy of phylogenetic analyses. To assess whether traits generally show phylogenetic associations, we present a quantitative review of 26 published phylogenetic comparative data sets. The data sets include 103 traits and were chosen from the ecological literature in which debate about the need for phylogenetic correction has been most acute. Eighty-eight percent of data sets contained at least one character that displayed significant phylogenetic dependence, and 60% of characters overall (pooled across studies) showed significant evidence of phylogenetic association. In 16% of tests, phylogenetic correlation could be neither supported nor rejected. However, most of these equivocal results were found in small phylogenies and probably reflect a lack of power. We suggest that the parameter lambda be routinely estimated when analyzing comparative data, since it can also be used simultaneously to adjust the phylogenetic correction in a manner that is optimal for the data set, and we present an example of how this may be done.

Delivery of active hexosaminidase across the blood-brain barrier in rats.

The ability to deliver enzymatically active human hexosaminidase A across the blood-brain barrier and into brain cells of the normal rat was examined. Following osmotic blood-brain barrier modification in the rat, intraarterially administered human hexosaminidase A and B were shown to cross the barrier and enter brain cells. Subcellular fractionation studies demonstrated that most of the human enzyme delivered across the barrier was functionally active and appeared to be inside a subcellular organelle. These studies provide evidence that blood-brain barrier modification permits delivery of functionally active hexosaminidase A into subcellular organelles consistent with that known to be the appropriate site of physiologic activity.

Variation across species in the size of the nuclear genome supports the junk-DNA explanation for the C-value paradox.

The amount of DNA in the nuclear genome (the DNA C-value) of eukaryotes varies at least 80,000-fold across species, and yet bears little or no relation to organismic complexity or to the number of protein-coding genes. This phenomenon is known as the C-value paradox. One explanation for the C-value paradox attributes the size of the nuclear genome to 'junk' (typically non-coding) genetic elements that accumulate until the costs to the organism of replicating excess DNA select against it. Across species, organisms that develop at a slower rate should tolerate more junk DNA. Alternatively, junk DNA may function as a nucleo-skeleton to maintain the volume of the nucleus at a size proportional to the volume of the cytoplasm in the cell. Across species, the DNA C-value is predicted to vary with the nuclear and cytoplasmic volumes of cells. Previous studies have not been able to distinguish between the skeletal-DNA and junk-DNA explanations for the C-value paradox. We report a study of DNA content in 24 salamander species which does. The size of the nuclear genome is correlated with developmental rate even after the effects of nuclear and cytoplasmic volume have been removed. However, genome size is not correlated with cytoplasmic volume after controlling for developmental rate. These results support the view that junk DNA accumulates in the nuclear genome until the costs of replicating it become too great, rather than that it functions as a nucleo-skeleton.

Further refinement of the Escherichia coli brain abscess model in rat.

The rat brain abscess model provides a substrate for the modeling of delivery of therapeutic agents to intracerebral mass lesions. We now report refinement of the Escherichia coli brain abscess model in rat. A K1 surface antigen-negative E. coli isolated from human blood culture was stereotaxically inoculated into deep brain sites. Histopathologic analyses and quantitative cultures demonstrated the consistent production of lesions. No animal in this consecutive series developed meningitis, ventriculitis or sepsis. By contrast, prior experience with E. coli abscess production resulted in 25% failure rate of abscess production or death from sepsis. This improvement in the model may be attributable to specific characteristics of the bacteria used, modification of the inoculation method or the intracerebral placement technique. The present work suggests a reliable and consistent brain abscess model, which may be further used to study brain suppuration.

White matter changes associated with feline GM2 gangliosidosis (Sandhoff disease): correlation of MR findings with pathologic and ultrastructural abnormalities.

PURPOSE: To establish changes on MR of the brain in a feline model of Sandhoff disease in order to develop standards by which this model may be used in future noninvasive studies. METHODS: Five affected felines and six age-matched, littermate controls were evaluated. T1- and T2-weighted images were obtained once or twice for each of four affected and five control animals at 4 1/2 to 12 weeks of age, for a total of 15 MR examinations. Images were evaluated qualitatively for the pattern of myelination and the size of the ventricular system. After the animals were killed, pathologic specimens of the brain were examined with light and electron microscopy, and pathologic changes were correlated with MR. RESULTS: Compared with control animals, affected animals showed MR evidence of delayed myelination, manifested by white matter signal hypointensity on T1-weighted images and signal hyperintensity on T2-weighted images. This finding was corroborated by histopathologic findings of decreased myelin in the subcortical and internal capsule regions. White matter abnormalities were not detected ultrastructurally in the animals examined. CONCLUSION: Although GM2 gangliosidosis is primarily a neuronal disease, MR imaging can show changes in myelination of white matter tracts that may be secondary to the neuronal damage. This provides a noninvasive method of in vivo monitoring as therapeutic strategies are developed in this animal model.

A physiological barrier distal to the anatomic blood-brain barrier in a model of transvascular delivery.

BACKGROUND AND PURPOSE: Osmotic disruption of the blood-brain barrier (BBB) provides a method for transvascular delivery of therapeutic agents to the brain. The apparent global delivery of viral-sized iron oxide particles to the rat brain after BBB opening as seen on MR images was compared with the cellular and subcellular location and distribution of the particles. METHODS: Two dextran-coated superparamagnetic monocrystalline iron oxide nanoparticle contrast agents, MION and Feridex, were administered intraarterially in rats at 10 mg Fe/kg immediately after osmotic opening of the BBB with hyperosmolar mannitol. After 2 to 24 hours, iron distribution in the brain was evaluated first with MR imaging then by histochemical analysis and electron microscopy to assess perivascular and intracellular distribution. RESULTS: After BBB opening, MR images showed enhancement throughout the disrupted hemisphere for both Feridex and MION. Feridex histochemical staining was found in capillaries of the disrupted hemisphere. Electron microscopy showed that the Feridex particles passed the capillary endothelial cells but did not cross beyond the basement membrane. In contrast, after MION delivery, iron histochemistry was detected within cell bodies in the disrupted hemisphere, and the electron-dense MION core was detected intracellularly and extracellularly in the neuropil. CONCLUSION: MR images showing homogeneous delivery to the brain at the macroscopic level did not indicate delivery at the microscopic level. These data support the presence of a physiological barrier at the basal lamina, analogous to the podocyte in the kidney, distal to the anatomic (tight junction) BBB, which may limit the distribution of some proteins and viral particles after transvascular delivery to the brain.