Exposure of Human Lung Cells to Polystyrene Microplastics Significantly Retards Cell Proliferation and Triggers Morphological Changes.

Microplastics in the environment produced by decomposition of globally increasing waste plastics have become a dominant component of both water and air pollution. To examine the potential toxicological effects of microplastics on human cells, the cultured human alveolar A549 cells were exposed to polystyrene microplastics (PS-MPs) of 1 and 10 μm diameter as a model of the environmental contaminants. Both sizes caused a significant reduction in cell proliferation but exhibited little cytotoxicity, as measured by the maintenance of cell viabilities determined by trypan blue staining and by Calcein-AM staining.

A Simple Adaptable Blood-Brain Barrier Cell Model for Screening Matrix Metalloproteinase Inhibitor Functionality.

The blood-brain barrier is a multicellular and basement membrane unit that regulates molecular transport between the blood and central nervous system. Many cerebral pathologies, such as acute stroke and chronic vascular dementia, result in a disrupted blood-brain barrier, increasing its permeability and allowing the entry of potentially neurotoxic molecules. The activation of matrix metalloproteinases mediates further blood-brain barrier damage.

A pseudo-plaque method for infectious particle assay and clonal isolation of adeno-associated virus.

A colorimetric method has been developed for the detection of adeno-associated virus (AAV) infectious centers in cell culture monolayers. Due to its non-cytopathic nature, AAV has not been amenable to the traditional plaque assay, involving an agar overlay and cellular stains. As a result, an alternate method was required.

Enhanced production and isotope enrichment of recombinant glycoproteins produced in cultured mammalian cells.

NMR studies of post-translationally modified proteins are complicated by the lack of an efficient method to produce isotope enriched recombinant proteins in cultured mammalian cells. We show that reducing the glucose concentration and substituting glutamate for glutamine in serum-free medium increased cell viability while simultaneously increasing recombinant protein yield and the enrichment of non-essential amino acids compared to culture in unmodified, serum-free medium. Adding dichloroacetate, a pyruvate dehydrogenase kinase inhibitor, further improves cell viability, recombinant protein yield, and isotope enrichment.

Twinned crystals of adeno-associated virus serotype 3b prove suitable for structural studies.

Adeno-associated viruses (AAVs) are leading candidate vectors for gene-therapy applications. The AAV-3b capsid is closely related to the well characterized AAV-2 capsid (87% identity), but sequence and presumably structural differences lead to distinct cell-entry and immune-recognition properties. In an effort to understand these differences and to perhaps harness them, diffraction-quality crystals of purified infectious AAV-3b particles have been grown and several partial diffraction data sets have been recorded.

Crystallization and preliminary X-ray structural studies of adeno-associated virus serotype 6.

Adeno-associated viruses are being developed as vectors for gene therapy and have been used in a number of clinical trials. Vectors to date have been based on the type species AAV-2, the structure of which was published in 2002. There is growing interest in modulating the cellular tropism and immune neutralization of AAV-2 with variants inspired by the properties of other serotypes.

Zinc deficiency impairs neuronal precursor cell proliferation and induces apoptosis via p53-mediated mechanisms.

The potential importance of stem cells in the adult central nervous system (CNS) that cannot only divide, but also participate in neurogenesis, is now widely appreciated. While we know that the trace element zinc is needed for brain development, the role of this essential nutrient in adult stem cell proliferation and neurogenesis has not been investigated. Adult male rats fed a zinc-restricted diet had approximately 50% fewer Ki67-positive stem cells in the subgranular zone (SGZ) and granular cell layer of the dentate gyrus compared to both zinc-adequate and pair-fed controls (p<0.

Curvature driven transport of mouse macrophages in a pulsating magnetic garnet film ratchet.

Magnetic fields varying on the colloidal length scale are used for the directed transport of magnetically labeled biological cells. The transport is achieved by using the ratchet effect which relies on an asymmetric, symmetry broken periodic potential where nonequilibrium fluctuations or oscillations generate a net cell current. Ferrofluid ingested mouse macrophages were placed on a magnetic garnet film with alternating stripe domain patterns, and a pulsating magnetic potential is provided by superposing an oscillating magnetic field normal to the film.

An economic approach to isotopic enrichment of glycoproteins expressed from Sf9 insect cells.

It is estimated that over half of all proteins are glycosylated, yet only a small number of the structures in the protein data bank are of intact glycoproteins. One of the reasons for the lack of structural information on glycoproteins is the high cost of isotopically labeling proteins expressed from eukaryotic cells such as in insect and mammalian cells. In this paper we describe modifications to commercial insect cell growth medium that reduce the cost for isotopically labeling recombinant proteins expressed from Sf9 cells.

Serotype-specific detection of adeno-associated virus during laboratory preparation.

Adeno-associated virus (AAV) is a small single-stranded DNA member of the family Parvoviradae with at least eight recognized human serotypes, several of which are being studied as candidate vectors for gene therapy. When multiple serotypes are handled in the same laboratory, it is critical to know the serotype of a sample with certainty. Here, a rapid and reliable PCR-based method is presented for the identification of serotypes 2, 3B or 6 and for screening for cross contamination.

Characterization of the capsid protein glycosylation of adeno-associated virus type 2 by high-resolution mass spectrometry.

Adeno-associated virus type 2 (AAV-2) capsid proteins have eight sequence motifs that are potential sites for O- or N-linked glycosylation. Three are in prominent surface locations, close to the sites of cellular receptor attachment and to neutralizing epitopes on or near protrusions surrounding the three-fold axes, raising the possibility that AAV-2 might use glycosylation as a means of immune escape or for preventing reattachment on release of progeny virus. Peptide mapping and structural analysis by Fourier transform ion cyclotron resonance mass spectrometry demonstrates, however, no glycosylation of the capsid protein for virus prepared in cultured HeLa cells.

Role of dietary iron restriction in a mouse model of Parkinson's disease.

There is a growing body of evidence suggesting that iron chelation may be a useful therapy in the treatment of Parkinson's Disease (PD). Experiments were designed to test the impact of dietary iron availability on the pathogenic process and functional outcome in a mouse model of PD. Mice were fed diets containing low (4 ppm) or adequate (48 ppm) amounts of iron for 6 weeks before the administration of MPTP, a mitochondrial toxin that damages nigrostriatal dopaminergic neurons and induces Parkinson-like symptoms.

Large-scale production, purification and crystallization of wild-type adeno-associated virus-2.

Adeno-associated virus-2 (AAV-2) has long been recognized as a potential vector for human gene therapy. Although much progress has been made in the molecular virology of AAV-2, structural studies of AAV-2 have been hampered by the low efficiency of virus production in culture, the low purity of preparations, and the low solubility of pure virus particles in solution. Methods of larger scale AAV-2 production have been developed through adaptation to suspension culture and re-optimization of the times of infection and transfection with respect to particle production.

Expression, purification, and characterization of avian Thy-1 from Lec1 mammalian and Tn5 insect cells.

Structural studies of asparagine-linked glycoproteins are complicated by the oligosaccharide heterogeneity inherent to individual glycosylation sites. Herein, we report the cloning of a novel isoform of avian Thy-1 and the subsequent expression, purification, and characterization of a soluble form of Thy-1 from Lec1 mammalian and Tn5 insect cells. The novel isoform of Thy-1 differs from the previously reported chicken isoform by eight amino acid residues, but these changes do not alter the secondary structure content, the disulfide bond pattern, or the sites of glycosylation.

The atomic structure of adeno-associated virus (AAV-2), a vector for human gene therapy.

The structure of the adeno-associated virus (AAV-2) has been determined to 3-A resolution by x-ray crystallography. AAV is being developed as a vector for gene therapy to treat diseases including hemophilia, cancer, and cystic fibrosis. As in the distantly related autonomous parvoviruses, the capsid protein has a beta-barrel fold, but long loops between the beta-strands share little structural homology with other parvoviruses, leading to unique surface features.