Biodegradable cationic poly(carbonates): effect of varying side chain hydrophobicity on key aspects of gene transfection

The degree of hydrophobicity in cationic polymers plays an important but often underappreciated role in the safety and efficacy of gene delivery processes. In order to further elucidate structure-activity relationships of biodegradable cationic poly(carbonate) gene carriers, we synthesized a series of narrowly dispersed homo-polymers via metal-free organocatalytic living ring-opening polymerization (ROP) of cyclic carbonate monomers bearing either alkyl (propyl, hexyl or nonyl) or 4-methyl benzyl halide side chains. The polymers were then quaternized using bis-tertiary amines to install both quaternary ammoniums and tertiary amines for DNA binding and endosomal escape, respectively. Among the polymers with similar molecular lengths and charge densities, it was found that an increase in side chain alkyl spacer length from 3 to 6 carbons significantly enhanced cellular uptake and luciferase gene expression in HepG2 and HeLa cell lines without causing overt hemolysis and cytotoxicity. A further increase of side chain alkyl length to 9 carbons, however, led to a drastic decline in gene expression due to increased cellular toxicity, which was correlated with an increased disruption and lysis of red blood cell membranes. Interestingly, the incorporation of an aromatic 4-methyl benzyl spacer increased DNA binding strength, reduced particle sizes of resultant DNA complexes, and enhanced cellular uptake, leading to improved luciferase gene expression, albeit with higher levels of hemolysis and cytotoxicity. Taken together, the findings of this study demonstrate that a delicate balance between cationic charge density and hydrophobicity could be achieved by utilizing a hexyl spacer in the side chains of cationic poly(carbonates), hence providing insights on the future development of non-viral cationic polymeric gene delivery systems. Statement of Significance: Owing to their ease of synthesis and well-controlled polymerization, biodegradable cationic poly(carbonates) have emerged as a highly promising class of biomaterials for gene delivery. The hydrophobicity of side chains in cationic polymers plays an important but often underappreciated role in influencing key aspects of gene transfection. In our efforts to improve gene transfection and understand structure-activity relationships, we synthesized a series of cationic polymers bearing a common poly(carbonate) backbone, and with side chains containing various hydrophobic spacers (propyl, hexyl, 4-methyl benzyl or nonyl) before the cationic moiety. A moderate degree of hydrophobicity was optimal as the cationic poly(carbonate) with hexyl side chains mediated high gene transfection efficiencies while causing low cytotoxicities. (111 words

Frozen and Invariant Quantum Discord under Local Dephasing Noise

In this chapter, we intend to explore and review some remarkable dynamical properties of quantum discord under various different open quantum system models. Specifically, our discussion will include several concepts connected to the phenomena of time invariant and frozen quantum discord. Furthermore, we will elaborate on the relation of these two phenomena to the non-Markovian features of the open system dynamics and to the usage of dynamical decoupling protocols.Comment: 29 pages, 8 figure

Broader Neutralizing Antibodies against H5N1 Viruses Using Prime-Boost Immunization of Hyperglycosylated Hemagglutinin DNA and Virus-Like Particles

BACKGROUND: Highly pathogenic avian influenza (HPAI) H5N1 viruses and their transmission capability from birds to humans have raised global concerns about a potential human pandemic. The inherent nature of antigenic changes in influenza viruses has not been sufficiently taken into account in immunogen designs for broadly protective HPAI H5N1 vaccines. METHODS: We designed a hyperglycosylated HA vaccine using N-linked glycan masking on highly variable sequences in the HA1 globular head. Immunization of these hyperglycosylated HA DNA vaccines followed by a flagellin-containing virus-like particle booster in mice was conducted to evaluate neutralizing antibody responses against various clades of HPAI H5N1 viruses. RESULTS: We introduced nine N-X-S/T motifs in five HA1 regions: 83NNT, 86NNT, 94NFT, 127NSS, 138NRT, 156NTT, 161NRS, 182NDT, and 252NAT according to sequence alignment analyses from 163 HPAI H5N1 human isolates. Although no significant differences of anti-HA total IgG titers were found with these hyperglycosyalted HA compared to the wild-type control, the 83NNT and 127NSS mutants elicited significantly potent cross-clade neutralizing antibodies against HPAI H5N1 viruses. CONCLUSIONS: This finding may have value in terms of novel immunogen design for developing cross-protective H5N1 vaccines

Dimethylarginine dimethylaminohydrolase I enhances tumour growth and angiogenesis

Angiogenesis is a prerequisite for tumour progression and is highly regulated by growth factors and cytokines a number of which also stimulate the production of nitric oxide. Asymmetric dimethylarginine is an endogenous inhibitor of nitric oxide synthesis. Asymmetric dimethylarginine is metabolised by dimethylarginine dimethylaminohydrolase. To study the effect of dimethylarginine dimethylaminohydrolase on tumour growth and vascular development, the rat C6 glioma cell line was manipulated to overexpress the rat gene for dimethylarginine dimethylaminohydrolase I. Enhanced expression of dimethylarginine dimethylaminohydrolase I increased nitric oxide synthesis (as indicated by a two-fold increase in the production of cGMP), expression and secretion of vascular endothelial cell growth factor, and induced angiogenesis in vitro. Tumours derived from these cells grew more rapidly in vivo than cells with normal dimethylarginine dimethylaminohydrolase I expression. Immunohistochemical and magnetic resonance imaging measurements were consistent with increased tumour vascular development. Furthermore, dimethylarginine dimethylaminohydrolase activity was detected in a series of human tumours. This data demonstrates that dimethylarginine dimethylaminohydrolase plays a pivotal role in tumour growth and the development of the tumour vasculature by regulating the concentration of nitric oxide and altering vascular endothelial cell growth factor production

Identification of DreI as an Antiviral Factor Regulated by RLR Signaling Pathway

BACKGROUND:Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) had been demonstrated to prime interferon (IFN) response against viral infection via the conserved RLR signaling in fish, and a novel fish-specific gene, the grass carp reovirus (GCRV)-induced gene 2 (Gig2), had been suggested to play important role in host antiviral response. METHODOLOGY/PRINCIPAL FINDINGS:In this study, we cloned and characterized zebrafish Gig2 homolog (named Danio rerio Gig2-I, DreI), and revealed its antiviral role and expressional regulation signaling pathway. RT-PCR, Western blot and promoter activity assay indicate that DreI can be induced by poly I:C, spring viremia of carp virus (SVCV) and recombinant IFN (rIFN), showing that DreI is a typical ISG. Using the pivotal signaling molecules of RLR pathway, including RIG-I, MDA5 and IRF3 from crucian carp, it is found that DreI expression is regulated by RLR cascade and IRF3 plays an important role in this regulation. Furthermore, promoter mutation assay confirms that the IFN-stimulated regulatory elements (ISRE) in the 5' flanking region of DreI is essential for its induction. Finally, overexpression of DreI leads to establish a strong antiviral state against SVCV and Rana grylio virus (RGV) infection in EPC (Epithelioma papulosum cyprinid) cells. CONCLUSIONS/SIGNIFICANCE:These data indicate that DreI is an antiviral protein, which is regulated by RLR signaling pathway

Exploring spatial-frequency-sequential relationships for motor imagery classification with recurrent neural network

Abstract Background Conventional methods of motor imagery brain computer interfaces (MI-BCIs) suffer from the limited number of samples and simplified features, so as to produce poor performances with spatial-frequency features and shallow classifiers. Methods Alternatively, this paper applies a deep recurrent neural network (RNN) with a sliding window cropping strategy (SWCS) to signal classification of MI-BCIs. The spatial-frequency features are first extracted by the filter bank common spatial pattern (FB-CSP) algorithm, and such features are cropped by the SWCS into time slices. By extracting spatial-frequency-sequential relationships, the cropped time slices are then fed into RNN for classification. In order to overcome the memory distractions, the commonly used gated recurrent unit (GRU) and long-short term memory (LSTM) unit are applied to the RNN architecture, and experimental results are used to determine which unit is more suitable for processing EEG signals. Results Experimental results on common BCI benchmark datasets show that the spatial-frequency-sequential relationships outperform all other competing spatial-frequency methods. In particular, the proposed GRU-RNN architecture achieves the lowest misclassification rates on all BCI benchmark datasets. Conclusion By introducing spatial-frequency-sequential relationships with cropping time slice samples, the proposed method gives a novel way to construct and model high accuracy and robustness MI-BCIs based on limited trials of EEG signals

Genomic analysis and temperature-dependent transcriptome profiles of the rhizosphere originating strain Pseudomonas aeruginosa M18

<p>Abstract</p> <p>Background</p> <p>Our previously published reports have described an effective biocontrol agent named <it>Pseudomonas </it>sp. M18 as its 16S rDNA sequence and several regulator genes share homologous sequences with those of <it>P. aeruginosa</it>, but there are several unusual phenotypic features. This study aims to explore its strain specific genomic features and gene expression patterns at different temperatures.</p> <p>Results</p> <p>The complete M18 genome is composed of a single chromosome of 6,327,754 base pairs containing 5684 open reading frames. Seven genomic islands, including two novel prophages and five specific non-phage islands were identified besides the conserved <it>P. aeruginosa </it>core genome. Each prophage contains a putative chitinase coding gene, and the prophage II contains a <it>capB </it>gene encoding a putative cold stress protein. The non-phage genomic islands contain genes responsible for pyoluteorin biosynthesis, environmental substance degradation and type I and III restriction-modification systems. Compared with other <it>P. aeruginosa </it>strains, the fewest number (3) of insertion sequences and the most number (3) of clustered regularly interspaced short palindromic repeats in M18 genome may contribute to the relative genome stability. Although the M18 genome is most closely related to that of <it>P. aeruginosa </it>strain LESB58, the strain M18 is more susceptible to several antimicrobial agents and easier to be erased in a mouse acute lung infection model than the strain LESB58. The whole M18 transcriptomic analysis indicated that 10.6% of the expressed genes are temperature-dependent, with 22 genes up-regulated at 28°C in three non-phage genomic islands and one prophage but none at 37°C.</p> <p>Conclusions</p> <p>The <it>P. aeruginosa </it>strain M18 has evolved its specific genomic structures and temperature dependent expression patterns to meet the requirement of its fitness and competitiveness under selective pressures imposed on the strain in rhizosphere niche.</p

Surface-Associated Plasminogen Binding of Cryptococcus neoformans Promotes Extracellular Matrix Invasion

BACKGROUND:The fungal pathogen Cryptococcus neoformans is a leading cause of illness and death in persons with predisposing factors, including: malignancies, solid organ transplants, and corticosteroid use. C. neoformans is ubiquitous in the environment and enters into the lungs via inhalation, where it can disseminate through the bloodstream and penetrate the central nervous system (CNS), resulting in a difficult to treat and often-fatal infection of the brain, called meningoencephalitis. Plasminogen is a highly abundant protein found in the plasma component of blood and is necessary for the degradation of fibrin, collagen, and other structural components of tissues. This fibrinolytic system is utilized by cancer cells during metastasis and several pathogenic species of bacteria have been found to manipulate the host plasminogen system to facilitate invasion of tissues during infection by modifying the activation of this process through the binding of plasminogen at their surface. METHODOLOGY:The invasion of the brain and the central nervous system by penetration of the protective blood-brain barrier is a prerequisite to the establishment of meningoencephalitis by the opportunistic fungal pathogen C. neoformans. In this study, we examined the ability of C. neoformans to subvert the host plasminogen system to facilitate tissue barrier invasion. Through a combination of biochemical, cell biology, and proteomic approaches, we have shown that C. neoformans utilizes the host plasminogen system to cross tissue barriers, providing support for the hypothesis that plasminogen-binding may contribute to the invasion of the blood-brain barrier by penetration of the brain endothelial cells and underlying matrix. In addition, we have identified the cell wall-associated proteins that serve as plasminogen receptors and characterized both the plasminogen-binding and plasmin-activation potential for this significant human pathogen. CONCLUSIONS:The results of this study provide evidence for the cooperative role of multiple virulence determinants in C. neoformans pathogenesis and suggest new avenues for the development of anti-infective agents in the prevention of fungal tissue invasion

Erythropoietin: a multimodal neuroprotective agent

The tissue protective functions of the hematopoietic growth factor erythropoietin (EPO) are independent of its action on erythropoiesis. EPO and its receptors (EPOR) are expressed in multiple brain cells during brain development and upregulated in the adult brain after injury. Peripherally administered EPO crosses the blood-brain barrier and activates in the brain anti-apoptotic, anti-oxidant and anti-inflammatory signaling in neurons, glial and cerebrovascular endothelial cells and stimulates angiogenesis and neurogenesis. These mechanisms underlie its potent tissue protective effects in experimental models of stroke, cerebral hemorrhage, traumatic brain injury, neuroinflammatory and neurodegenerative disease. The preclinical data in support of the use of EPO in brain disease have already been translated to first clinical pilot studies with encouraging results with the use of EPO as a neuroprotective agent