Microfluidic synthesis of monodisperse hierarchical silica particles with raspberry-like morphology

A novel and facile microfluidic approach is presented to transform silica precursor droplets into uniform hierarchical microparticles with raspberry-like surface morphology. This novel microfluidic method provides a new strategy to make microparticles with unique surface morphology, and could open new opportunities for potential applications in catalysis, energy, biomedical fields

Combined In-Fermenter Extraction and Cross-Flow Microfiltration for Improved Inclusion Body Processing

In this study we demonstrate a new in-fermenter chemical extraction procedure that degrades the cell wall of Escherichia coli and releases inclusion bodies (IBs) into the fermentation medium. We then prove that cross-flow microfiltration can be used to remove 91% of soluble contaminants from the released IBs. The extraction protocol, based on a combination of Triton X-100, EDTA, and intracellular T7 lysozyme, effectively released most of the intracellular soluble content without solubilising the IBs. Cross-flow microfiltration using a 0.2 m ceramic membrane successfully recovered the granulocyte macrophage-colony stimulating factor (GM-CSF) IBs with removal of 91% of the soluble contaminants and virtually no loss of IBs to the permeate. The filtration efficiency, in terms of both flux and transmission, was significantly enhanced by in-fermenter Benzonase® digestion of nucleic acids following chemical extraction. Both the extraction and filtration methods exerted their efficacy directly on a crude fermentation broth, eliminating the need for cell recovery and resuspension in buffer. The processes demonstrated here can all be performed using just a fermenter and a single cross-flow filtration unit, demonstrating a high level of process intensification. Furthermore, there is considerable scope to also use the microfiltration system to subsequently solubilise the IBs, to separate the denatured protein from cell debris, and to refold the protein using diafiltration. In this way refolded protein can potentially be obtained, in a relatively pure state, using only two unit operations

Non-Carrier Nanoparticles Adjuvant ModularProtein Vaccine in a Particle-Dependent Manner

Nanoparticles are increasingly used to adjuvant vaccine formulations due to their biocompatibility, ease of manufacture and the opportunity to tailor their size, shape, and physicochemical properties. The efficacy of similarly-sized silica (Si-OH), poly (D,L-lactic-co-glycolic acid) (PLGA) and poly caprolactone (PCL) nanoparticles (nps) to adjuvant recombinant capsomere presenting antigenic M2e modular peptide from Influenza A virus (CapM2e) was investigated in vivo. Formulation of CapM2e with Si-OH or PLGA nps significantly boosted the immunogenicity of modular capsomeres, even though CapM2e was not actively attached to the nanoparticles prior to injection (i.e., formulation was by simple mixing). In contrast, PCL nps showed no significant adjuvant effect using this simple-mixing approach. The immune response induced by CapM2e alone or formulated with nps was antibody-biased with very high antigen-specific antibody titer and less than 20 cells per million splenocytes secreting interferon gamma. Modification of silica nanoparticle surface properties through amine functionalization and pegylation did not lead to significant changes in immune response. This study confirms that simple mixing-based formulation can lead to effective adjuvanting of antigenic protein, though with antibody titer dependent on nanoparticle physicochemical properties

Hybrid organic-inorganic nanoparticles: controlled incorporation of gold nanoparticles into virus-like particles and application in surface-enhanced Raman spectroscopy.

A capsid is the protein coat surrounding a virus' genome that ensures its protection and transport. The capsid of murine polyomavirus (muPy) consists of one major (VP1) and two minor (VP2/3) proteins, from which just VP1 is sufficient to form the capsid when expressed recombinantly (1). From a material engineering point of view, viral capsids are of interest because they present a paradigm for complex self-assembly on the nanometer scale. Understanding and controlling these assembly dynamics will allow the construction of nanoscale structures using a self-assembly process. The first step in this direction was the discovery that capsids of several viruses can be reversibly disassembled into their building blocks and reassembled using the same building blocks by simply changing the buffer conditions (2, 3). Such capsids already find applications as targeted in vivo delivery vectors for genes, proteins or small molecular drugs (4, 5), as optical probes for biomedical imaging and sensing purposes with unprecedented resolution and sensitivity and can potentially be used as templates for nanoelectronics (6, 7)

Controlled generation of ultrathin-shell double emulsions and studies on their stability

Double emulsions with a hierarchical core-shell structure have great potential in various applications, but their broad use is limited by their instability. To improve stability, water-in-oil-in-water (W/O/W) emulsions with an ultrathin oil layer of several hundred nanometres were produced by using a microcapillary device. The effects of various parameters on the generation of ultrathin-shell double emulsions and their droplet size were investigated, including the proper combinations of inner, middle and outer phases, flow rates and surfactants. The surfactant in the middle oil phase was found to be critical for the formation of the ultrathin-shell double emulsions. Furthermore, the stability of these double emulsions can be notably improved by increasing the concentration of the surfactant, and they can be stable for months. This opens up new opportunities for their future applications in cosmetics, foods and pharmaceuticals

Deep ATLAS radio observations of the CDFS-SWIRE field

We present the first results from the Australia Telescope Large Area Survey (ATLAS), which consist of deep radio observations of a 3.7 square degree field surrounding the Chandra Deep Field South, largely coincident with the infrared Spitzer Wide-Area Extragalactic (SWIRE) Survey. We also list cross-identifications to infrared and optical photometry data from SWIRE, and ground-based optical spectroscopy. A total of 784 radio components are identified, corresponding to 726 distinct radio sources, nearly all of which are identified with SWIRE sources. Of the radio sources with measured redshifts, most lie in the redshift range 0.5-2, and include both star-forming galaxies and active galactic nuclei (AGN). We identify a rare population of infrared-faint radio sources which are bright at radio wavelengths but are not seen in the available optical, infrared, or X-ray data. Such rare classes of sources can only be discovered in wide, deep surveys such as this.Comment: Accepted by A

Genome-wide meta-analysis of 241,258 adults accounting for smoking behaviour identifies novel loci for obesity traits

Few genome-wide association studies (GWAS) account for environmental exposures, like smoking, potentially impacting the overall trait variance when investigating the genetic contribution to obesity-related traits. Here, we use GWAS data from 51,080 current smokers and 190,178 nonsmokers (87% European descent) to identify loci influencing BMI and central adiposity, measured as waist circumference and waist-to-hip ratio both adjusted for BMI. We identify 23 novel genetic loci, and 9 loci with convincing evidence of gene-smoking interaction (GxSMK) on obesity-related traits. We show consistent direction of effect for all identified loci and significance for 18 novel and for 5 interaction loci in an independent study sample. These loci highlight novel biological functions, including response to oxidative stress, addictive behaviour, and regulatory functions emphasizing the importance of accounting for environment in genetic analyses. Our results suggest that tobacco smoking may alter the genetic susceptibility to overall adiposity and body fat distribution.Peer reviewe

Preparative protein refolding

The rapid provision of purified native protein underpins both structural biology and the development of new biopharmaceuticals. The dominance of Escherichia coli as a cellular biofactory depends on technology for solubilizing and refolding, proteins, that are expressed as insoluble inclusion bodies. Such technology must be scale invariant, easily automated, generic for a broad range of similar proteins and economical. Refolding methods relying on denaturant dilution and column-based approaches meet these criteria. Recent developments, particularly in column-based methods, promise to extend the range of proteins that can be refolded successfully. Developments in preparing denatured purified protein and in the analysis of protein refolding products promise to remove bottlenecks in the overall process. Combined, these developments promise to facilitate the rapid and automated determination of appropriate refolding conditions and to simplify scale-up

Large-scale recovery of protein inclusion bodies by continuous centrifugation

Inclusion bodies (IBs) are micron-sized solid protein particles that form within the cytoplasm of certain host cells such as Escherichia coli following overexpression of a protein. IBs are comprised primarily of the recombinant protein of interest. Some contaminants including nonproduct protein, nucleic acids, and cell-envelope contaminants can also be incorporated into the granules. However, it is believed that the majority of contaminants actually adhere to the IB surface following release from the cytoplasm during processing (1). This indicates that IB formation in vivo is a rather specific process that offers certain advantages for downstream processing. Specifically, the protein of interest already exists in a relatively pure state as a small granule that can be recovered by physical separation from nonassociated contaminants. Of course, if a suitably efficient protein refolding strategy is not available (2), then any gains achieved through inclusion body formation may be easily lost

Non-infectious virus-like particles for the validation of membrane integrity and column performance in bioprocessing

Effective viral clearance is critical in both water treatment and biopharmaceutical manufacture to protect public health. In this study, virus-like particle (VLP) was used as a surrogate for membrane and column performance assessments. A reliable ELISA method was developed to quantify VLPs with high sensitivity and specificity. Log reduction value (LRV) was measured in a scaled-down manner by mimicking the unit operation using VLPs. This study provides a potential to utilise VLPs in the real-time validation on membrane and column performance in water-treatment and biopharmaceutical manufacturing industries