3D Primary Hepatocyte Culture Systems for Analyses of Liver Diseases,Drug Metabolism,and Toxicity: Emerging Culture Paradigms and Applications

Recent research has shown that the maintenance of relevant liver functions ex vivo requires models in which the cells exhibit an in vivo‐like phenotype, often achieved by reconstitution of appropriate cellular interactions. Multiple different models have been presented that differ in the cells utilized, media, and culture conditions. Furthermore, several technologically different approaches have been presented including bioreactors, chips, and plate‐based systems in fluidic or static media constituting of chemically diverse materials. Using such models, the ability to predict drug metabolism, drug toxicity, and liver functionality have increased tremendously as compared to conventional in vitro models in which cells are cultured as 2D monolayers. Here, the authors highlight important considerations for microphysiological systems for primary hepatocyte culture, review current culture paradigms, and discuss their opportunities for studies of drug metabolism, hepatotoxicity, liver biology, and disease.     

Complex Formation between Two Biosynthetic Enzymes Modifies the Allosteric Regulatory Properties of Both: AN EXAMPLE OF MOLECULAR SYMBIOSIS*

Allostery, where remote ligand binding alters protein function, is essential for the control of metabolism. Here, we have identified a highly sophisticated allosteric response that allows complex control of the pathway for aromatic amino acid biosynthesis in the pathogen Mycobacterium tuberculosis. This response is mediated by an enzyme complex formed by two pathway enzymes: chorismate mutase (CM) and 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS). Whereas both enzymes are active in isolation, the catalytic activity of both enzymes is enhanced, and in particular that of the much smaller CM is greatly enhanced (by 120-fold), by formation of a hetero-octameric complex between CM and DAH7PS. Moreover, on complex formation M. tuberculosis CM, which has no allosteric response on its own, acquires allosteric behavior to facilitate its own regulatory needs by directly appropriating and partly reconfiguring the allosteric machinery that provides a synergistic allosteric response in DAH7PS. Kinetic and analytical ultracentrifugation experiments demonstrate that allosteric binding of phenylalanine specifically promotes hetero-octameric complex dissociation, with concomitant reduction of CM activity. Together, DAH7PS and CM from M. tuberculosis provide exquisite control of aromatic amino acid biosynthesis, not only controlling flux into the start of the pathway, but also directing the pathway intermediate chorismate into either Phe/Tyr or Trp biosynthesis.     

Mutations in CKAP2L,the Human Homolog of the Mouse Radmis Gene,Cause Filippi Syndrome

Filippi syndrome is a rare, presumably autosomal-recessive disorder characterized by microcephaly, pre- and postnatal growth failure, syndactyly, and distinctive facial features, including a broad nasal bridge and underdeveloped alae nasi. Some affected individuals have intellectual disability, seizures, undescended testicles in males, and teeth and hair abnormalities. We performed homozygosity mapping and whole-exome sequencing in a Sardinian family with two affected children and identified a homozygous frameshift mutation, c.571dupA (p.Ile191Asnfs^∗6), in CKAP2L, encoding the protein cytoskeleton-associated protein 2-like (CKAP2L). The function of this protein was unknown until it was rediscovered in mice as Radmis (radial fiber and mitotic spindle) and shown to play a pivotal role in cell division of neural progenitors. Sanger sequencing of CKAP2L in a further eight unrelated individuals with clinical features consistent with Filippi syndrome revealed biallelic mutations in four subjects. In contrast to wild-type lymphoblastoid cell lines (LCLs), dividing LCLs established from the individuals homozygous for the c.571dupA mutation did not show CKAP2L at the spindle poles. Furthermore, in cells from the affected individuals, we observed an increase in the number of disorganized spindle microtubules owing to multipolar configurations and defects in chromosome segregation. The observed cellular phenotypes are in keeping with data from in vitro and in vivo knockdown studies performed in human cells and mice, respectively. Our findings show that loss-of-function mutations in CKAP2L are a major cause of Filippi syndrome.     

Stress inhibits psychomotor performance differently in simple and complex open field environments

Stress affects psychomotor profiles and exploratory behavior in response to environmental features. Here we investigated psychomotor and exploratory patterns induced by stress in a simple open-field arena and a complex, multi-featured environment. Groups of rats underwent seven days of restraint stress or no-stress conditions and were individually tested in three versions of the ziggurat task (ZT) that varied according to environmental complexity. The hyperactivity of the hypothalamic–pituitary–adrenal (HPA) axis due to stress procedure was evaluated by the pre- and post-stress levels of circulating corticosterone (CORT). Horizontal activity, exploration, and motivation were measured by the number of fields entered, the time spent in the central fields, path length and speed, and stop duration. In addition, vertical exploratory behavior was measured by the times rats climbed onto ziggurats. Stress-induced psychomotor changes were indicated by reduced path length and path speed and increased duration of stops only within the complex arena of the ZT. Rats in stress groups also showed a significant decline in the vertical movements as measured by the number of climbing onto ziggurats. No stress-induced changes were revealed by the simple open-field arena. The exploratory patterns of stressed animals suggest psychomotor inhibition and reduced novelty-seeking behaviors in an environment-dependent manner. Thus, multi-featured arenas that require complex behavioral strategies are ideally suited to reveal the inhibitory effects of stress on psychomotor capabilities in rodents.     

Correlation between body size and fatty acid and essential amino acid composition of round goby (Neogobius melanostomus) and monkey goby (Neogobius fluviatilis) from the Rhine River (Germany)

In this study correlations between body size and muscle fatty and amino acid content of two species of goby, round goby (Neogobius melanostomus) and monkey goby (Neogobius fluviatilis) caught from river Rhine (Germany) were investigated. Among saturated fatty acids (SFAs), mono- (MUFA) and polyunsaturated fatty acids (PUFAs) only SFAs were significantly higher in round goby than monkey goby (P < 0.05). In general, the correlation between body size of both gobies and the content of most of the individual fatty acids was not significant. In monkey goby, the content of palmitic acid (C16:0) and oleic acid (C18:1 n-9) was positively correlated with weight (r = 0.43) and total length (r = ?0.58), respectively, and the content of docosahexaenoic acid (DHA) increased with condition factor (r = 0.50). The content of threonine, arginine, valine, phenylalanine and isoleucine in monkey goby was higher than those of round goby (P < 0.05). In round goby the three essential amino acids arginine, valine and leucine were positively (P < 0.05) correlated with body length, which indicates that longer round gobies are of higher nutritional value.     

Measuring Microtubule Polarity in Spindles with Second-Harmonic Generation

The spatial organization of microtubule polarity, and the interplay between microtubule polarity and protein localization, is thought to be crucial for spindle assembly, anaphase, and cytokinesis, but these phenomena remain poorly understood, in part due to the difficulty of measuring microtubule polarity in spindles. We develop and implement a method to nonperturbatively and quantitatively measure microtubule polarity throughout spindles using a combination of second-harmonic generation and two-photon fluorescence. We validate this method using computer simulations and by comparison to structural data on spindles obtained from electron tomography and laser ablation. This method should provide a powerful tool for studying spindle organization and function, and may be applicable for investigating microtubule polarity in other systems.     

Structure of the Hemoglobin-IsdH Complex Reveals the Molecular Basis of Iron Capture by Staphylococcus aureus

Staphylococcus aureus causes life-threatening disease in humans. The S. aureus surface protein iron-regulated surface determinant H (IsdH) binds to mammalian hemoglobin (Hb) and extracts heme as a source of iron, which is an essential nutrient for the bacteria. However, the process of heme transfer from Hb is poorly understood. We have determined the structure of IsdH bound to human Hb by x-ray crystallography at 4.2 Å resolution, revealing the structural basis for heme transfer. One IsdH molecule is bound to each α and β Hb subunit, suggesting that the receptor acquires iron from both chains by a similar mechanism. Remarkably, two near iron transporter (NEAT) domains in IsdH perform very different functions. An N-terminal NEAT domain binds α/β globin through a site distant from the globin heme pocket and, via an intervening structural domain, positions the C-terminal heme-binding NEAT domain perfectly for heme transfer. These data, together with a 2.3 Å resolution crystal structure of the isolated N-terminal domain bound to Hb and small-angle x-ray scattering of free IsdH, reveal how multiple domains of IsdH cooperate to strip heme from Hb. Many bacterial pathogens obtain iron from human hemoglobin using proteins that contain multiple NEAT domains and other domains whose functions are poorly understood. Our results suggest that, rather than acting as isolated units, NEAT domains may be integrated into higher order architectures that employ multiple interaction interfaces to efficiently extract heme from host proteins.     

Measurement of in vivo cerebral volumetric strain induced by the Valsalva maneuver

Compressibility of biological tissues such as brain parenchyma is related to its poroelastic nature characterized by the geometry and pressure of vasculature and interconnected fluid-filled spaces. Thus, cerebral volumetric strain may be sensitive to intracranial pressure which can be altered under physiological conditions. So far volumetric strain has attained little attention in studies of the mechanical behavior of the brain.     

Silicon nutrition potentiates the antioxidant metabolism of rice plants under iron toxicity

Iron toxicity reduces growth of rice plants in acidic lowlands. Silicon nutrition may alleviate many stresses including heavy metal toxicity in plants. In the present study, the ameliorating effects of silicon nutrition on rice (Oryza sativa L.) plants under toxic Fe levels were investigated. Plants were cultivated in greenhouse in hydroponics under different Fe treatments including 10, 50, 100, and 250 mg L^?1 as Fe-EDTA and silicon nutrition including 0 and 1.5 mM sodium silicate. Iron toxicity imposed significant reduction in plant fresh weight, tiller, and leaf number. The activity of catalase, cell wall, and soluble peroxidases, and polyphenol oxidase in shoots decreased due to moderate Fe toxicity (50 and 100 mg L^?1), but increased at greater Fe concentration. Ascorbate peroxidase activity increased in both roots and shoots of Fe-stressed plants. Iron toxicity led to increased tissue hydrogen peroxide and lipid peroxidation. Silicon nutrition improved plant growth under all Fe treatments and alleviated Fe toxicity symptoms, probably due to lower Fe concentration of Si-treated plants. Silicon application could improve the activity of antioxidant enzymes such as catalase, ascorbate peroxidase, and soluble peroxidase under moderate Fe toxicity, which resulted in greater hydrogen peroxide detoxification and declined lipid peroxidation. Thus, silicon nutrition could ameliorate harmful effects of Fe toxicity possibly through reduction of plant Fe concentration and improvement of antioxidant enzyme activity.     

An ice nucleation protein from Fusarium acuminatum: cloning,expression, biochemical characterization and computational modeling

Ice nucleation proteins (INP) are a major cause of frost damage in plants and crops. Here, an INP gene from Fusarium acuminatum was optimized, synthesized, expressed in E.coli and subsequently purified and characterized. The protein belongs to the second class of ice nucleation proteins with an optimum pH 5.5, relative activity and stability between pH 5 and 9.5 and up to 45 °C. The protein was fully active and stable in the presence of dimethyl sulfoxide (DMSO), dioxane, acetone and ethyl acetate. Moreover, it retained over 50 % of its original activity in the presence of polyvinyl alcohol. The 3D structure model of the INP-F indicated the protein had three distinct domains as exist in other ice nucleation proteins with some variations. Considering these promising results, INP-F could be a novel candidate for industrial applications.