Microtubule regulation of N-methyl-D-aspartate receptor channels in neurons

N-Methyl-D-aspartate (NMDA) receptors (NMDARs), which play a key role in synaptic plasticity, are dynamically regulated by many signaling molecules and scaffolding proteins. Although actin cytoskeleton has been implicated in regulating NMDAR stability in synaptic membrane, the role of microtubules in regulating NMDAR trafficking and function is largely unclear. Here we show that microtubule-depolymerizing agents inhibited NMDA receptor-mediated ionic and synaptic currents in cortical pyramidal neurons. This effect was Ca(2+)-independent, required GTP, and was more prominent in the presence of high NMDA concentrations. The NR2B subunit-containing NMDA receptor was the primary target of microtubules. The effect of microtubule depolymerizers on NMDAR currents was blocked by cellular knockdown of the kinesin motor protein KIF17, which transports NR2B-containing vesicles along microtubule in neuronal dendrites. Neuromodulators that can stabilize microtubules, such as brain-derived neurotrophic factor, significantly attenuated the microtubule depolymerizer-induced reduction of NMDAR currents. Moreover, immunocytochemical studies show that microtubule depolymerizers decreased the number of surface NR2B subunits on dendrites, which was prevented by the microtubule stabilizer. Taken together, these results suggest that interfering with microtubule assembly suppresses NMDAR function through a mechanism dependent on kinesin-based dendritic transport of NMDA receptors.     

CD36-deficient mice are resistant to alcohol- and high-carbohydrate-induced hepatic steatosis

CD36 is a scavenger receptor with multiple ligands and cellular functions, including facilitating cellular uptake of free fatty acids (FFAs). Chronic alcohol consumption increases hepatic CD36 expression, leading to the hypothesis that this promotes uptake of circulating FFAs, which then serve as a substrate for triglyceride (TG) synthesis and the development of alcoholic steatosis. We investigated this hypothesis in alcohol-fed wild-type and Cd36-deficient (Cd36^−/−) mice using low-fat/high-carbohydrate Lieber-DeCarli liquid diets, positing that Cd36^−/− mice would be resistant to alcoholic steatosis. Our data show that the livers of Cd36^−/− mice are resistant to the lipogenic effect of consuming high-carbohydrate liquid diets. These mice also do not further develop alcoholic steatosis when chronically fed alcohol. Surprisingly, we did not detect an effect of alcohol or CD36 deficiency on hepatic FFA uptake; however, the lower baseline levels of hepatic TG in Cd36^−/− mice fed a liquid diet were associated with decreased expression of genes in the de novo lipogenesis pathway and a lower rate of hepatic de novo lipogenesis. In conclusion, Cd36^−/− mice are resistant to hepatic steatosis when fed a high-carbohydrate liquid diet, and they are also resistant to alcoholic steatosis. These studies highlight an important role for CD36 in hepatic lipid homeostasis that is not associated with hepatic fatty acid uptake.     

Separation and enrichment of neural stem cells using segregation in an expanded bed

An expanded bed system has been developed for a novel application in which the separation and enrichment of neural stem cells from a sample containing a mixture of stem and progenitor cells is achieved based on the difference in the sizes of the aggregates of these types of cells. Inert Sephadex beads and flocculated yeast cells were used as experimental controls and references. The characteristics of the separation of neural stem cell aggregates based on size are similar to those achieved with flocculated yeast where cell-to-cell aggregation controls the pattern of size separation different from those of inert Sephadex beads.     

Isolation of Laribacter hongkongensis, a novel bacterium associated with gastroenteritis, from drinking water reservoirs in Hong Kong

AIMS: Freshwater fish has been found to be the reservoir of Laribacter hongkongensis, a recently discovered bacterium associated with community-acquired gastroenteritis. However, little is known about the ecology of this bacterium in the aquatic environment. We carried out a surveillance study to investigate the presence of L. hongkongensis in water and freshwater fish from 10 drinking water reservoirs in Hong Kong. METHODS AND RESULTS: Using membrane filtration, L. hongkongensis was isolated from the waters of six reservoirs, with numbers ranging from 1 to 12 CFU l(-1). Higher recovery rates were observed in summer and during days of higher water and ambient temperatures. Of 27 freshwater fish collected from the reservoirs, L. hongkongensis was recovered from the intestines of two fish, a Goldfish and a Nile tilapia. Overall, 35 different pulsed-field gel electrophoresis patterns are found among the 59 isolates recovered from water and the two isolates from freshwater fish. CONCLUSIONS: The present report represents the first to demonstrate the presence of L. hongkongensis in natural water environments. SIGNIFICANCE AND IMPACT OF THE STUDY: Although it is unlikely that treated, drinking water is an important source of L. hongkongensis-associated gastroenteritis, one should be aware of the possibility of other contaminated water as a source of human infection.     

A humanized neutralizing antibody against MERS-CoV targeting the receptor-binding domain of the spike protein

The newly-emerging Middle East respiratory syndrome coronavirus (MERS-CoV) can cause severe and fatal acute respiratory disease in humans. Despite global efforts, the potential for an associated pandemic in the future cannot be excluded. The development of effective counter-measures is urgent. MERS-CoV-specific anti-viral drugs or vaccines are not yet available. Using the spike receptor-binding domain of MERS-CoV (MERS-RBD) to immunize mice, we identified two neutralizing monoclonal antibodies (mAbs) 4C2 and 2E6. Both mAbs potently bind to MERS-RBD and block virus entry in vitro with high efficacy. We further investigated their mechanisms of neutralization by crystallizing the complex between the Fab fragments and the RBD, and solved the structure of the 4C2 Fab/MERS-RBD complex. The structure showed that 4C2 recognizes an epitope that partially overlaps the receptor-binding footprint in MERS-RBD, thereby interfering with the virus/receptor interactions by both steric hindrance and interface-residue competition. 2E6 also blocks receptor binding, and competes with 4C2 for binding to MERS-RBD. Based on the structure, we further humanized 4C2 by preserving only the paratope residues and substituting the remaining amino acids with the counterparts from human immunoglobulins. The humanized 4C2 (4C2h) antibody sustained similar neutralizing activity and biochemical characteristics to the parental mouse antibody. Finally, we showed that 4C2h can significantly abate the virus titers in lungs of Ad5-hCD26-transduced mice infected with MERS-CoV, therefore representing a promising agent for prophylaxis and therapy in clinical settings.     

Inhibition of Rhizoctonia by endospore forming bacteria indigenous to landscape planting beds

Endospore forming bacteria were collected from root samples of 35 genera of bedding plants growing in established commercial landscape beds in Central Florida. One hundred and twenty-nine bacterial strains associated with 14 species were identified using fatty acid analysis (Microbial Identification System, MIDI). All strains were evaluated for in vitro inhibition of damping off disease caused by the fungus Rhizoctonia solani. The strongest inhibition of Rhizoctonia by soluble exudates in cocultivation was observed with strains belonging to six species: B. cereus, B. pumilus, B. thuringiensis, L. sphaericus, B. amyloliquefaciens, and B. subtilis. Most strains that inhibited Rhizoctonia growth in cocultivation also inhibited growth via volatile compounds. All 129 strains were evaluated for ability to protect impatiens plants from subsequent challenge infection by Rhizoctonia solani. Certain strains of endospore forming bacteria also enhanced plant growth. It is apparent from this study that the Bacillus community associated with bedding plants in established planting beds produce soluble antifungal compounds, volatile antifungal compounds, and enhance plant growth. In developing biological control, it may be a more practical approach to promote or enhance a natural, multifaceted community of Bacillus strains within our planting beds.     

Glycan analysis of glycoprotein pharmaceuticals: Evaluation of analytical approaches to Z number determination in pharmaceutical erythropoietin products

N-Glycosylation of many glycoprotein drugs is important for biological activity and should therefore be the target of specific and quantitative analytical methods. In this study, we focus on the two N-glycan mapping approaches that are used in pharmacopoeial monograph to analyse N-glycans released from fifteen preparations of recombinant human erythropoietin supplied by ten Chinese manufacturers. Underivatised N-glycans were analysed by high performance anion-exchange chromatography with pulsed amperometric detection and fluorophore-labelled N-glycans were analysed by weak anion-exchange and normal-phase high performance liquid chromatography. N-glycans were also analysed by matrix assisted laser desorption ionisation mass spectrometry. The release of N-glycans by PNGase F was shown to be consistent. Z number, a mathematical expression of the total negatively charged N-glycans composition has provided a convenient way to summarise the complex dataset and it might be suitable for product consistency monitoring. However, this Z number reduces the information of individual acidic N-glycan structure and is also found to be method dependent. Therefore, its use requires clear specification and validation. In this study, we only found weak but positive correlation between the Z number and its bioactivity. Wide range of N-glycans yields were obtained from the fifteen preparations but the significance of their differences is unclear.