Flow Rate and Apparent Volume of Cerebrospinal Fluid in Rhesus Macaques (Macaca mulatta) Based on the Pharmacokinetics of Intrathecally Administered Inulin

Cerebrospinal fluid (CSF) flow rate and volume are fundamental to the design and interpretation of preclinical pharmacokinetics and pharmacodynamics studies in NHP. To determine the values of CSF flow rate and volume, we evaluated the plasma and CSF pharmacokinetics of inulin, an inert polysaccharide tracer, in 5 rhesus macaques with CSF ventricular reservoirs and lumbar ports; these reservoirs and ports facilitate humane intrathecal administration and serial CSF sampling in unanesthetized macaques. Inulin was administered intrathecally via the CSF ventricular reservoir (n = 3), followed by the collection of lumbar CSF via the lumbar port and plasma. The contribution of dietary inulin was evaluated by using pre- and postprandial inulin plasma concentrations (n = 2) and a feed analysis of the NHP diet. Inulin concentrations were quantified using ELISA. Pharmacokinetic parameters were calculated by using noncompartmental methods. Daily diet was analyzed for inulin by using Official Method no. 997.08 of AOAC International. In male rhesus macaques, the mean CSF flow rate, established via inulin clearance after IT administration, was 0.018 ± 0.003 mL/min; mean CSF volume, established based on apparent volume of distribution, was 10.17 ± 0.63 mL. In plasma, inulin was quantifiable in all pre-administration samples and increased over the sampling period, precluding interpretation of plasma pharmacokinetics. Evaluation of the effect of diet on plasma concentrations established quantifiable inulin levels that showed minimal variation relative to the prandial state. Analysis of the feed detected 5 inulin types ranging from 1100 to 1440 mg per100 g. The diet was the source of detectable pre-administration inulin plasma concentrations, whereas inulin was not detected in CSF before inulin administration.

Successful treatment of CNS disease with a therapeutic agent requires CNS penetration of the agent across the blood–brain barrier (BBB) to its site of action, to achieve an effective concentration and duration.³⁰ The BBB⁷ limits access into the CNS for systemically administered agents through a complex physical and chemical system.²⁸ In the presence of CNS disease such as malignant glioma, the properties of the BBB may undergo changes permitting greater diffusion of systemically administered agents into the CNS.²⁸ However, the BBB is not static, and permeability changes in the presence of disease may be transient, occur only partially, or not occur.³⁰ Therefore, an agent''s activity against a target demonstrated in vitro, or in vivo in preclinical murine animal models, may be ineffective in patient clinical trials, when the agent fails to reach the target or does not reach the target at an effective concentration or duration^17,27 due to the varying, potentially restrictive, permeability of the BBB in patients. Intrathecal (IT) drug administration is an established alternative administration route that bypasses the BBB and delivers the agent directly into the CSF.²⁹ IT administration is accomplished as intraventricular delivery into the ventricles of the brain or as intralumbar delivery into the spinal column.Efficacious treatment regimens for systemic or IT administration of an agent fundamentally rely on preclinical pharmacokinetics–pharmacodynamics studies that provide information on parameters such as drug plasma and CSF concentrations, duration of measurable drug (pharmacokinetics) or drug activity (pharmacodynamics), drug elimination and distribution, and adverse events.^22,26 Drug exposure, or concentration of a drug over time, is defined by the Area Under the Curve, (AUC). The duration is typically described as the elimination half-life (t_1/2), which is the time required for half of the agent to be biologically reduced quantitatively. Elimination is represented by clearance, which is defined as the rate at which an agent is biologically removed. Distribution, as the apparent volume of distribution (V_d), is the apparent fluid volume required to contain the total amount of drug administered as it relates to the drug concentration in the biologic fluid (plasma, serum, whole blood, or CSF) from which it was measured.¹The interpretation of pharmacokinetics parameters, such as AUC, t_1/2, clearance, and V_d, to develop IT treatment rationales is improved when the species-specific values of CSF flow rate and volume are available for comparison to preclinical pharmacokinetics study results. Uniquely, the values of CSF volume and flow rate are independent of body weight,¹¹ and they serve to establish the potential concentration of drug in the CSF space (exposure and duration) and a mechanism for clearance via either CSF flow or absorption (elimination and distribution).Previously established NHP CSF access models—the CSF ventricular reservoir¹² and lumbar port¹⁶ models (Figure 1)—were developed in combination and used with inulin to evaluate rhesus macaque CSF flow rate and volume. These NHP models, developed in our laboratory, facilitate humane systemic and IT administration (via the CSF ventricular reservoir for the current study) as well as rapid serial CSF collection (via the lumbar port) and plasma collection, via an indwelling femoral intravenous port, in unanesthetized rhesus macaques. Inulin, a plant-based water-soluble polysaccharide, is relatively unaffected by absorption or secretion and is resistant to degradation allowing the substance to be used as a tracer in biologic fluid.^4,19 Because these properties of inulin preclude diffusion across the BBB and tissue absorption, a flow rate and volume can be calculated by using a known administered quantity.Open in a separate windowFigure 1.NHP CSF access models. CSF ventricular (lateral and 4^th) reservoirs and lumbar port.In the current study, we determined the CSF flow rate via clearance and of volume via V_d (hereafter as apparent volume) in rhesus macaques after intraventricular administration of inulin, lumbar CSF collection, and the subsequent quantification and pharmacokinetics analysis of the agent in CSF. Plasma concentrations of inulin were determined also. Because inulin was found to be quantifiable in the plasma prior to intraventricular administration for the pharmacokinetics study, we performed a secondary study to analyze the daily feed as a potential source of inulin in the plasma and to determine the influence of the daily NHP diet on pre- and postprandial plasma inulin levels.     

Arabidopsis ATG4 cysteine proteases specificity toward ATG8 substrates

Macroautophagy (hereafter autophagy) is a regulated intracellular process during which cytoplasmic cargo engulfed by double-membrane autophagosomes is delivered to the vacuole or lysosome for degradation and recycling. Atg8 that is conjugated to phosphatidylethanolamine (PE) during autophagy plays an important role not only in autophagosome biogenesis but also in cargo recruitment. Conjugation of PE to Atg8 requires processing of the C-terminal conserved glycine residue in Atg8 by the Atg4 cysteine protease. The Arabidopsis plant genome contains 9 Atg8 (AtATG8a to AtATG8i) and 2 Atg4 (AtATG4a and AtATG4b) family members. To understand AtATG4’s specificity toward different AtATG8 substrates, we generated a unique synthetic substrate C-AtATG8-ShR (citrine-AtATG8-Renilla luciferase SuperhRLUC). In vitro analyses indicated that AtATG4a is catalytically more active and has broad AtATG8 substrate specificity compared with AtATG4b. Arabidopsis transgenic plants expressing the synthetic substrate C-AtAtg8a-ShR is efficiently processed by endogenous AtATG4s and targeted to the vacuole during nitrogen starvation. These results indicate that the synthetic substrate mimics endogenous AtATG8, and its processing can be monitored in vivo by a bioluminescence resonance energy transfer (BRET) assay. The synthetic Atg8 substrates provide an easy and versatile method to study plant autophagy during different biological processes.     

Diversity and ecology of algae from the Nahal Qishon river,northern Israel

In 59 samples of periphyton and phytoplankton collected in 2002 - 2003 from the Nahal Qishon (Qishon River), northern Israel, we found 178 species from seven divisions of algae and cyanoprocaryotes. Diatoms, clorophytes, and cyanoprocaryotes prevail. Nitzschia and Navicula (Bacillariophyta) are the most abundant. Most of the species are cosmopolitan or widespread, except Lagynion janei (Chrysophyta), which is endemic for the Mediterranean Realm. About 17% of species (26) are new for Israel and five of them represent the first recorded genera: Crinalium endophyticum Crow, Actinocyclus normanii (Gregory) Hustedt, Rhizoclonium hieroglyphicum (Agardh) Kütz (Chlorophyta), Lagynion janei Bourelly, and Stylococcus aureus Chodat. Most of them come from a rare riverine assemblage with red alga Audouinella pygmea, as well as from the estuarine assemblage. Alkaliphiles predominate among the indicators of acidity, with few acidophiles confined to the communities under the impact of industrial wastes. Among the indicators of salinity, most numerous are the oligohalobien-indifferents and species adapted to a moderate salinity level. The relative species richness of ecological groups and the indices of saprobity are correlated with changes in conductivity, pH, and N-nitrate concentration. Indicators of organic pollution fall in the range of betameso- to alfamesosaprobic self-purification grades. Our studies show ecological significance of the Nahal Qishon as a model for a strongly disturbed aquatic ecosystem in the coastal zone of eastern Mediterranean.     

Responses of maize roots to drying – limits of viability

Turgid pieces of mature maize roots were dried in air and progressive changes in their relative water content (RWC) determined. Viability was tested by reproducibility of the drying curves after dehydration to successively lower RWCs. After reaching a chosen RWC, the pieces were rehydrated (approximately 2 h), and a 2nd and 3rd dehydration curve measured. Each drying curve was characterized by two parameters (a scale parameter λ, and a shape parameter β) of a survivorship function, which is a linear function of time. The parameter λ is more informative, and does not change in successive dehydrations for RWC > 0·4, suggesting no irreversible damage to the roots. Damage and death were indicated by divergences of λ in successive dehydrations to RWC = 0·35–0·15. Cryo-analytical microscopy confirmed these data while indicating specifically death of 50 and 100% of cortical cells at RWC 0·30 and 0·15, respectively, and survival of 50% or more of sieve tubes, pericycle and vascular parenchyma cells at root RWC as low as 0·15. This pattern of stelar cell survival may allow roots to preserve their capacity for renewal of axial conductivity and branch root development following periods of severe water stress.     

A Dynamic Hydrophobic Core and Surface Salt Bridges Thermostabilize a Designed Three-Helix Bundle

Thermostable proteins are advantageous in industrial applications, as pharmaceuticals or biosensors, and as templates for directed evolution. As protein-design methodologies improve, bioengineers are able to design proteins to perform a desired function. Although many rationally designed proteins end up being thermostable, how to intentionally design de novo, thermostable proteins is less clear. UVF is a de novo-designed protein based on the backbone structure of the Engrailed homeodomain (EnHD) and is highly thermostable (T_m > 99°C vs. 52°C for EnHD). Although most proteins generally have polar amino acids on their surfaces and hydrophobic amino acids buried in their cores, protein engineers followed this rule exactly when designing UVF. To investigate the contributions of the fully hydrophobic core versus the fully polar surface to UVF’s thermostability, we built two hybrid, chimeric proteins combining the sets of buried and surface residues from UVF and EnHD. Here, we determined a structural, dynamic, and thermodynamic explanation for UVF’s thermostability by performing 4 μs of all-atom, explicit-solvent molecular dynamics simulations at 25 and 100°C, Tanford-Kirkwood solvent accessibility Monte Carlo electrostatic calculations, and a thermodynamic analysis of 40 temperature runs by the weighted-histogram analysis method of heavy-atom, structure-based models of UVF, EnHD, and both chimeric proteins. Our models showed that UVF was highly dynamic because of its fully hydrophobic core, leading to a smaller loss of entropy upon folding. The charged residues on its surface made favorable electrostatic interactions that contributed enthalpically to its thermostability. In the chimeric proteins, both the hydrophobic core and charged surface independently imparted thermostability.     

Molecular phylogeny of Neotropical bioluminescent beetles (Coleoptera: Elateroidea) in southern and central Brazil

Bioluminescence in beetles is found mainly in the Elateroidea superfamily (Elateridae, Lampyridae and Phengodidae). The Neotropical region accounts for the richest diversity of bioluminescent species in the world with about 500 described species, most occurring in the Amazon, Atlantic rainforest and Cerrado (savanna) ecosystems in Brazil. The origin and evolution of bioluminescence, as well as the taxonomic status of several Neotropical taxa in these families remains unclear. In order to contribute to a better understanding of the phylogeny and evolution of bioluminescent Elateroidea we sequenced and analyzed sequences of mitochondrial NADH2 and the nuclear 28S genes and of the cloned luciferase sequences of Brazilian species belonging to the following genera: (Lampyridae) Macrolampis, Photuris, Amydetes, Bicellonycha, Aspisoma, Lucidota, Cratomorphus; (Elateridae) Conoderus, Pyrophorus, Hapsodrilus, Pyrearinus, Fulgeochlizus; and (Phengodidae) Pseudophengodes, Phrixothrix, Euryopa and Brasilocerus. Our study supports a closer phylogenetic relationship between Elateridae and Phengodidae as other molecular studies, in contrast with previous morphologic and molecular studies that clustered Lampyridae/Phengodidae. Molecular data also supported division of the Phengodinae subfamily into the tribes Phengodini and Mastinocerini. The position of the genus Amydetes supports the status of the Amydetinae as a subfamily. The genus Euryopa is included in the Mastinocerini tribe within the Phengodinae/Phengodidae. Copyright © 2013 John Wiley & Sons, Ltd.     

Insights into the structural nature of the transition state in the Kir channel gating pathway

In a previous study we identified an extensive gating network within the inwardly rectifying Kir1.1 (ROMK) channel by combining systematic scanning mutagenesis and functional analysis with structural models of the channel in the closed, pre-open and open states. This extensive network appeared to stabilize the open and pre-open states, but the network fragmented upon channel closure. In this study we have analyzed the gating kinetics of different mutations within key parts of this gating network. These results suggest that the structure of the transition state (TS), which connects the pre-open and closed states of the channel, more closely resembles the structure of the pre-open state. Furthermore, the G-loop, which occurs at the center of this extensive gating network, appears to become unstructured in the TS because mutations within this region have a ‘catalytic’ effect upon the channel gating kinetics.     

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator’s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 10¹⁰ viable and respiration competent mitochondria from 0.18 ± 0.04 g (wet weight) tissue sample.     

Structural and Functional Consequences of the Cardiac Troponin C L48Q Ca(2+)-Sensitizing Mutation

Calcium binding to the regulatory domain of cardiac troponin C (cNTnC) causes a conformational change that exposes a hydrophobic surface to which troponin I (cTnI) binds, prompting a series of protein-protein interactions that culminate in muscle contraction. A number of cTnC variants that alter the Ca(2+) sensitivity of the thin filament have been linked to disease. Tikunova and Davis engineered a series of cNTnC mutations that altered Ca(2+) binding properties and studied the effects on the Ca(2+) sensitivity of the thin filament and contraction [Tikunova, S. B., and Davis, J. P. (2004) J. Biol. Chem. 279, 35341-35352]. One of the mutations they engineered, the L48Q variant, resulted in a pronounced increase in the cNTnC Ca(2+) binding affinity and Ca(2+) sensitivity of cardiac muscle force development. In this work, we sought structural and mechanistic explanations for the increased Ca(2+) sensitivity of contraction for the L48Q cNTnC variant, using an array of biophysical techniques. We found that the L48Q mutation enhanced binding of both Ca(2+) and cTnI to cTnC. Nuclear magnetic resonance chemical shift and relaxation data provided evidence that the cNTnC hydrophobic core is more exposed with the L48Q variant. Molecular dynamics simulations suggest that the mutation disrupts a network of crucial hydrophobic interactions so that the closed form of cNTnC is destabilized. The findings emphasize the importance of cNTnC's conformation in the regulation of contraction and suggest that mutations in cNTnC that alter myofilament Ca(2+) sensitivity can do so by modulating Ca(2+) and cTnI binding.