Development of a novel purification method for AAV vectors using tangential flow filtration.

Adeno-associated virus (AAV) vector can efficiently transduce therapeutic genes in various tissue types with less side effects; however, owing to complex multistep processes during manufacture, there have been surges in the pricing of recently approved AAV vector-based gene therapy products. This study aimed to develop a simple and efficient method for high-quality purification of AAV vector via tangential flow filtration (TFF), which is commonly used for concentration and diafiltration of solutions during AAV vector purification. We established a novel purification method using TFF and surfactants. Treatment with two classes of surfactants (anionic and zwitterionic) successfully inhibited the aggregation of residual proteins separated from the AAV vector in the crude product by TFF, obtaining a clearance of 99.5% residual proteins. Infectivity of the AAV vector purified using the new method was confirmed both in vitro and in vivo, and no remarkable inflammation or tissue damage was observed in mouse skeletal muscle after local administration. Overall, our proposed method could be used to establish a platform for the purification of AAV vector.

Targeted single-cell genomics reveals novel host adaptation strategies of the symbiotic bacteria Endozoicomonas in Acropora tenuis coral.

BACKGROUND: Endozoicomonas bacteria symbiosis with various marine organisms is hypothesized as a potential indicator of health in corals. Although many amplicon analyses using 16S rRNA gene have suggested the diversity of Endozoicomonas species, genome analysis has been limited due to contamination of host-derived sequences and difficulties in culture and metagenomic analysis. Therefore, the evolutionary and functional potential of individual Endozoicomonas species symbiotic with the same coral species remains unresolved. RESULTS: In this study, we applied a novel single-cell genomics technique using droplet microfluidics to obtain single-cell amplified genomes (SAGs) for uncultured coral-associated Endozoicomonas spp. We obtained seven novel Endozoicomonas genomes and quantitative bacterial composition from Acropora tenuis corals at four sites in Japan. Our quantitative 16S rRNA gene and comparative genomic analysis revealed that these Endozoicomonas spp. belong to different lineages (Clade A and Clade B), with widely varying abundance among individual corals. Furthermore, each Endozoicomonas species possessed various eukaryotic-like genes in clade-specific genes. It was suggested that these eukaryotic-like genes might have a potential ability of different functions in each clade, such as infection of the host coral or suppression of host immune pathways. These Endozoicomonas species may have adopted different host adaptation strategies despite living symbiotically on the same coral. CONCLUSIONS: This study suggests that coral-associated Endozoicomonas spp. on the same species of coral have different evolutional strategies and functional potentials in each species and emphasizes the need to analyze the genome of each uncultured strain in future coral-Endozoicomonas relationships studies. Video Abstract.

Single-cell metabolite detection and genomics reveals uncultivated talented producer.

The production of bioactive metabolites is increasingly recognized as an important function of host-associated bacteria. An example is defensive symbiosis that might account for much of the chemical richness of marine invertebrates including sponges (Porifera), 1 of the oldest metazoans. However, most bacterial members of sponge microbiomes have not been cultivated or sequenced, and therefore, remain unrecognized. Unequivocally linking metabolic functions to a cellular source in sponge microbiomes is, therefore, a challenge. Here, we report an analysis pipeline of microfluidic encapsulation, Raman microscopy, and integrated digital genomics (MERMAID) for an efficient identification of uncultivated producers. We applied this method to the chemically rich bacteriosponge (sponge that hosts a rich bacterial community) Theonella swinhoei, previously shown to contain 'Entotheonella' symbionts that produce most of the bioactive substances isolated from the sponge. As an exception, the antifungal aurantosides had remained unassigned to a source. Raman-guided single-bacterial analysis and sequencing revealed a cryptic, distinct multiproducer, 'Candidatus Poriflexus aureus' from a new Chloroflexi lineage as the aurantoside producer. Its exceptionally large genome contains numerous biosynthetic loci and suggested an even higher chemical richness of this sponge than previously appreciated. This study highlights the importance of complementary technologies to uncover microbiome functions, reveals remarkable parallels between distantly related symbionts of the same host, and adds functional support for diverse chemically prolific lineages being present in microbial dark matter.

Molecular profiling of lipid droplets inside HuH7 cells with Raman micro-spectroscopy.

Raman imaging has become an attractive technology in molecular biology because of its ability to detect multiple molecular components simultaneously without labeling. Two major limitations in accurately accounting for spectral features, viz., background removal and spectral unmixing, have been overcome by employing a modified and effective routine in multivariate curve resolution (MCR). With our improved strategy, we have spectrally isolated seven structurally specific biomolecules without any post-acquisition spectral treatments. Consequently, the isolated intensity profiles reflected concentrations of corresponding biomolecules with high statistical accuracy. Our study reveals the changes in the molecular composition of lipid droplets (LDs) inside HuH7 cells and its relation to the physiological state of the cell. Further, we show that the accurate separation of spectral components permits analysis of structural modification of molecules after cellular uptake. A detailed discussion is presented to highlight the potential of Raman spectroscopy with MCR in semi-quantitative molecular profiling of living cells.

Rapid inspection method for investigating the heat processing conditions employed for chicken meat using Raman spectroscopy.

In Japan, the imports of meat products have been increasing every year. Heat processing of meat is the current standard method for ensuring domestic animal health, particularly in case of meat products from areas where infectious diseases are known to have occurred in domestic animals. The Animal Quarantine Service needs to establish a method that detects the temperature at which the meat has been heat-processed (endpoint temperature) to ensure that the standard protocol is followed at the production location. Here, we developed a Raman spectroscopy and multivariate statistics (viz. multivariate curve resolution (MCR))-based simple and rapid method for accurately estimating the end point temperature. We showed that the temperature-dependent secondary structure modification of proteins can serve as an accurate indicator of the temperature of heat processing. This methodology can be easily automated for effective utilization by someone who is not an expert in spectroscopy. We envisage a wider application of this method in food analysis, although the present research investigated the application of this method in chicken meat heat processing analysis.

In situ detection of antibiotic amphotericin B produced in Streptomyces nodosus using Raman microspectroscopy.

The study of spatial distribution of secondary metabolites within microbial cells facilitates the screening of candidate strains from marine environments for functional metabolites and allows for the subsequent assessment of the production of metabolites, such as antibiotics. This paper demonstrates the first application of Raman microspectroscopy for in situ detection of the antifungal antibiotic amphotericin B (AmB) produced by actinomycetes-Streptomyces nodosus. Raman spectra measured from hyphae of S. nodosus show the specific Raman bands, caused by resonance enhancement, corresponding to the polyene chain of AmB. In addition, Raman microspectroscopy enabled us to monitor the time-dependent change of AmB production corresponding to the growth of mycelia. The Raman images of S. nodosus reveal the heterogeneous distribution of AmB within the mycelia and individual hyphae. Moreover, the molecular association state of AmB in the mycelia was directly identified by observed Raman spectral shifts. These findings suggest that Raman microspectroscopy could be used for in situ monitoring of antibiotic production directly in marine microorganisms with a method that is non-destructive and does not require labeling.