Adeno-associated Virus (AAV) Serotypes Have Distinctive Interactions with Domains of the Cellular AAV Receptor.

Adeno-associated virus (AAV) entry is determined by its interactions with specific surface glycans and a proteinaceous receptor(s). Adeno-associated virus receptor (AAVR) (also named KIAA0319L) is an essential cellular receptor required for the transduction of vectors derived from multiple AAV serotypes, including the evolutionarily distant serotypes AAV2 and AAV5. Here, we further biochemically characterize the AAV-AAVR interaction and define the domains within the ectodomain of AAVR that facilitate this interaction. By using a virus overlay assay, it was previously shown that the major AAV2 binding protein in membrane preparations of human cells corresponds to a glycoprotein with a molecular mass of 150 kDa. By establishing a purification procedure, performing further protein separation by two-dimensional electrophoresis, and utilizing mass spectrometry, we now show that this glycoprotein is identical to AAVR. While we find that AAVR is an N-linked glycosylated protein, this glycosylation is not a strict requirement for AAV2 binding or functional transduction. Using a combination of genetic complementation with deletion constructs and virus overlay assays with individual domains, we find that AAV2 functionally interacts predominantly with the second Ig-like polycystic kidney disease (PKD) repeat domain (PKD2) present in the ectodomain of AAVR. In contrast, AAV5 interacts primarily through the first, most membrane-distal, PKD domain (PKD1) of AAVR to promote transduction. Furthermore, other AAV serotypes, including AAV1 and -8, require a combination of PKD1 and PKD2 for optimal transduction. These results suggest that despite their shared dependence on AAVR as a critical entry receptor, different AAV serotypes have evolved distinctive interactions with the same receptor.IMPORTANCE Over the past decade, AAV vectors have emerged as leading gene delivery tools for therapeutic applications and biomedical research. However, fundamental aspects of the AAV life cycle, including how AAV interacts with host cellular factors to facilitate infection, are only partly understood. In particular, AAV receptors contribute significantly to AAV vector transduction efficiency and tropism. The recently identified AAV receptor (AAVR) is a key host receptor for multiple serotypes, including the most studied serotype, AAV2. AAVR binds directly to AAV2 particles and is rate limiting for viral transduction. Defining the AAV-AAVR interface in more detail is important to understand how AAV engages with its cellular receptor and how the receptor facilitates the entry process. Here, we further define AAV-AAVR interactions, genetically and biochemically, and show that different AAV serotypes have discrete interactions with the Ig-like PKD domains of AAVR. These findings reveal an unexpected divergence of AAVR engagement within these parvoviruses.

Cytotoxic activity of Holothuria tubulosa (Echinodermata) coelomocytes.

The immune system of marine invertebrates, in particular that of holothurians, still requires further study. Our research showed that coelomocyte cells contained in the coelomic fluid of the sea cucumber, Holothuria tubulosa, are able to lyse, in vitro, red blood cells in rabbits and sheep. A plaque-forming assay showed spherule cells to be the effector cells, able to release cytotoxic molecules after xenogenic cell contact. The coelomocyte lysate supernatant, analysed by polyacrylamide gel electrophoresis overlay technique, using rabbit and sheep erythrocytes, showed two different haemolytic protein patterns: one calcium dependent and the other calcium independent. The fractions of each pattern were resolved on a polyacrylamide gel and calcium-dependent and independent coelomocyte lysate patterns were compared.

Evaluating Nanoparticulate Vaccine Formulations for Effective Antigen Presentation and T-Cell Proliferation Using an In Vitro Overlay Assay.

Inducing T lymphocyte (T-cell) activation and proliferation with specificity against a pathogen is crucial in vaccine formulation. Assessing vaccine candidates' ability to induce T-cell proliferation helps optimize formulation for its safety, immunogenicity, and efficacy. Our in-house vaccine candidates use microparticles (MPs) and nanoparticles (NPs) to enhance antigen stability and target delivery to antigen-presenting cells (APCs), providing improved immunogenicity. Typically, vaccine formulations are screened for safety and immunostimulatory effects using in vitro methods, but extensive animal testing is often required to assess immunogenic responses. We identified the need for a rapid, intermediate screening process to select promising candidates before advancing to expensive and time-consuming in vivo evaluations. In this study, an in vitro overlay assay system was demonstrated as an effective high-throughput preclinical testing method to evaluate the immunogenic properties of early-stage vaccine formulations. The overlay assay's effectiveness in testing particulate vaccine candidates for immunogenic responses has been evaluated by optimizing the carboxyfluorescein succinimidyl ester (CFSE) T-cell proliferation assay. DCs were overlaid with T-cells, allowing vaccine-stimulated DCs to present antigens to CFSE-stained T-cells. T-cell proliferation was quantified using flow cytometry on days 0, 1, 2, 4, and 6 upon successful antigen presentation. The assay was tested with nanoparticulate vaccine formulations targeting Neisseria gonorrhoeae (CDC F62, FA19, FA1090), measles, H1N1 flu prototype, canine coronavirus, and Zika, with adjuvants including Alhydrogel® (Alum) and AddaVax™. The assay revealed robust T-cell proliferation in the vaccine treatment groups, with variations between bacterial and viral vaccine candidates. A dose-dependent study indicated immune stimulation varied with antigen dose. These findings highlight the assay's potential to differentiate and quantify effective antigen presentation, providing valuable insights for developing and optimizing vaccine formulations.

Phosphatidic acid and phosphoinositides facilitate liposome association of Yas3p and potentiate derepression of ARE1 (alkane-responsive element one)-mediated transcription control.

In the n-alkane assimilating yeast Yarrowia lipolytica, the expression of ALK1, encoding a cytochrome P450 that catalyzes terminal mono-oxygenation of n-alkanes, is induced by n-alkanes. The transcription of ALK1 is regulated by a heterocomplex that comprises the basic helix-loop-helix transcription activators, Yas1p and Yas2p, and binds to alkane-responsive element 1 (ARE1) in the ALK1 promoter. An Opi1 family transcription repressor, Yas3p, represses transcription by binding to Yas2p. Yas3p localizes in the nucleus when Y. lipolytica is grown on glucose but localizes to the endoplasmic reticulum (ER) upon the addition of n-alkanes. In this study, we showed that recombinant Yas3p binds to the acidic phospholipids, phosphatidic acid (PA) and phosphoinositides (PIPs), in vitro. The ARE1-mediated transcription was enhanced in vivo in mutants defective in an ortholog of the Saccharomyces cerevisiae gene PAH1, encoding PA phosphatase, and in an ortholog of SAC1, encoding PIP phosphatase in the ER. Truncation mutation analyses for Yas3p revealed two regions that bound to PA and PIPs. These results suggest that the interaction with acidic phospholipids is important for the n-alkane-induced association of Yas3p with the ER membrane.

Optimisation of an agar overlay assay for the assessment of the antimicrobial activity of topically applied semi-solid antiseptic products including honey-based formulations.

The aim of this study was to optimise the so-called agar overlay assay to investigate the antimicrobial activity of some currently available topical antimicrobial products against a range of Gram positive and Gram negative bacteria. During the optimisation process, various assay parameters including base agar volume, overlay agar volume, overlay agar concentration, placement of products into wells or onto coverslip and inoculum concentration were taken into consideration. The optimised assay was found to be a convenient, suitable and effective platform for the assessment of the antimicrobial activity of commercial semi-solid over-the-counter (OTC) products (i.e. non-prescription medicines for topical application used commonly without supervision by a healthcare professional) containing a range of active pharmaceutical ingredients and to be potentially also applicable to complex natural product-based formulations (e.g. honey-based formulation, melaleuca oil-based formulation). The most auspicious aspect of the optimised method was its capability of determining the antimicrobial activity of intact products without further manipulation, unlike other tests that require products to be dissolved or dilute, thus compromising their integrity.

Cell-Free Protein Translation System for Expression of Lipid-Binding Proteins Tagged with Small epitopes and Their Use in Protein-Lipid Overlay Assays.

We adapted an efficient cell-free protein synthesis-based protocol for the production of lipid-binding proteins. The experimental procedures are based on the following steps: (1) cell-free synthesis of soluble, lipid-binding proteins fused to small tags; (2) analysis by dot blot of the accessibility of antibodies to the small tags. (3) protein lipid overlay assay with, immunodetection of bound protein by either chemiluminescence or fluorescence. We also provide a fast and inexpensive protocol for homemade lipid nitrocellulose strips spotted with acidic lipids (mostly phosphoinositides) extracted from plant tissues. These homemade lipid strips can be used for preliminary screen and characterization of putative phosphoinositide-binding proteins.

Bacteriophage Enumeration and Detection Methods.

Application of phages as alternative antimicrobials to combat pathogenic bacteria and their association to a healthy gut microbiome has prompted a need for precise methods for detection and enumeration of phage particles. There are many applicable methods, but care should be taken considering the measured object (infectious phage, whole phage particle or nucleic acid and proteins) and the concept behind the technique to avoid misinterpretations. While molecular methods cannot discriminate between viable and non-infectious phages, the traditional techniques for counting infectious phages can be time consuming and poorly reproducible. Here, we describe the methods currently used for phage detection and enumeration and highlight their advantages as well as their limitations. Finally, we provide insight on how to deal with complex samples, as well as future prospects in the field of phage quantification.

The Integration of Genome Mining, Comparative Genomics, and Functional Genetics for Biosynthetic Gene Cluster Identification.

Antimicrobial resistance is a worldwide health crisis for which new antibiotics are needed. One strategy for antibiotic discovery is identifying unique antibiotic biosynthetic gene clusters that may produce novel compounds. The aim of this study was to demonstrate how an integrated approach that combines genome mining, comparative genomics, and functional genetics can be used to successfully identify novel biosynthetic gene clusters that produce antimicrobial natural products. Secondary metabolite clusters of an antibiotic producer are first predicted using genome mining tools, generating a list of candidates. Comparative genomic approaches are then used to identify gene suites present in the antibiotic producer that are absent in closely related non-producers. Gene sets that are common to the two lists represent leading candidates, which can then be confirmed using functional genetics approaches. To validate this strategy, we identified the genes responsible for antibiotic production in Pantoea agglomerans B025670, a strain identified in a large-scale bioactivity survey. The genome of B025670 was first mined with antiSMASH, which identified 24 candidate regions. We then used the comparative genomics platform, EDGAR, to identify genes unique to B025670 that were not present in closely related strains with contrasting antibiotic production profiles. The candidate lists generated by antiSMASH and EDGAR were compared with standalone BLAST. Among the common regions was a 14 kb cluster consisting of 14 genes with predicted enzymatic, transport, and unknown functions. Site-directed mutagenesis of the gene cluster resulted in a reduction in antimicrobial activity, suggesting involvement in antibiotic production. An integrated approach that combines genome mining, comparative genomics, and functional genetics yields a powerful, yet simple strategy for identifying potentially novel antibiotics.

Breast Cancer Cells Transition from Mesenchymal to Amoeboid Migration in Tunable Three-Dimensional Silk-Collagen Hydrogels.

Invading cancer cells adapt their migration phenotype in response to mechanical and biochemical cues from the extracellular matrix. For instance, mesenchymal migration is associated with strong cell-matrix adhesions and an elongated morphology, while amoeboid migration is associated with minimal cell-matrix adhesions and a rounded morphology. However, it remains challenging to elucidate the role of matrix mechan-ics and biochemistry, since these are both dependent on ECM protein concentration. Here, we demonstrate a composite silk fibroin and collagen I hydrogel where stiffness and microstructure can be systematically tuned over a wide range. Using an overlay assay geometry, we show that the invasion of metastatic breast cancer cells exhibits a biphasic dependence on silk fibroin concentration at fixed collagen I concentration, first increasing as the hydrogel stiffness increases, then decreasing as the pore size of silk fibroin decreases. Indeed, mesenchymal morphology exhibits a similar biphasic depen-dence on silk fibroin concentration, while amoeboid morphologies were favored when cell-matrix adhesions were less effective. We used exogenous biochemical treatment to perturb cells towards increased contractility and a mesenchymal morphology, as well as to disrupt cytoskeletal function and promote an amoeboid morphology. Overall, we envision that this tunable biomaterial platform in a 96-well plate format will be widely applicable to screen cancer cell migration against combinations of designer biomaterials and targeted inhibitors.

Quantitative Assessment of Antimicrobial Activity of PLGA Films Loaded with 4-Hexylresorcinol.

Profound screening and evaluation methods for biocide-releasing polymer films are crucial for predicting applicability and therapeutic outcome of these drug delivery systems. For this purpose, we developed an agar overlay assay embedding biopolymer composite films in a seeded microbial lawn. By combining this approach with model-dependent analysis for agar diffusion, antimicrobial potency of the entrapped drug can be calculated in terms of minimum inhibitory concentrations (MICs). Thus, the topical antiseptic 4-hexylresorcinol (4-HR) was incorporated into poly(lactic-co-glycolic acid) (PLGA) films at different loadings up to 3.7 mg/cm² surface area through a solvent casting technique. The antimicrobial activity of 4-HR released from these composite films was assessed against a panel of Gram-negative and Gram-positive bacteria, yeasts and filamentous fungi by the proposed assay. All the microbial strains tested were susceptible to PLGA-4-HR films with MIC values down to 0.4% (w/w). The presented approach serves as a reliable method in screening and quantifying the antimicrobial activity of polymer composite films. Moreover, 4-HR-loaded PLGA films are a promising biomaterial that may find future application in the biomedical and packaging sector.

Isolation of ferritin and its interaction with BmNPV in the silkworm, Bombyx mori.

Ferritin is a ubiquitous iron storage protein that plays an important role in host defence against pathogen infections. In the present study, native ferritin was isolated from the hemolymph of Bombyx mori using native-polyacrylamide gel electrophoresis (native-PAGE) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results revealed that ferritin consisted of two subunits, designated as BmFerHCH and BmFerLCH. Previously integrated previous transcriptome and iTRAQ data showed that the two subunits were down-regulated in resistant silkworm strain BC9 and there was no obvious change in the expression levels of the subunits in susceptible silkworm strain P50 after BmNPV infection. Virus overlay assays revealed that B. mori ferritin as the form of heteropolymer had an interaction with B. mori nucleopolyhedrovirus (BmNPV), but it can't interact with BmNPV after depolymerisation. What's more, reverse transcription quantitative PCR (RT-qPCR) analysis suggested that BmFerHCH and BmFerLCH could be induced by bacteria, virus and iron. This is the first study to extract B. mori ferritin successfully and confirms their roles in the process of BmNPV infection. All these results will lay a foundation for further research the function of B. mori ferritin.

Bacterial Amyloids.

Many bacteria can assemble functional amyloid fibers on their cell surface. Most bacterial amyloids contribute to biofilm or other community behaviors where cells interact with a surface or with other cells. Bacterial amyloids, like all functional amyloids, share structural and biochemical properties with disease-associated eukaryotic amyloids. The general ability of amyloids to bind specific dyes, like Congo red and Thioflavin T, and their resistance to denaturation have provided useful tools for scoring and quantifying bacterial amyloid formation. Here, we present basic approaches to study bacterial amyloids by focusing on the well-studied curli amyloid fibers expressed by Enterobacteriaceae. These methods exploit the specific tinctorial and biophysical properties of amyloids. The methods described here are straightforward and can be easily applied by any modern molecular biology lab for the study of other bacterial amyloids.

Identification of Lipid Binding Modulators Using the Protein-Lipid Overlay Assay.

The protein-lipid overlay assay is an inexpensive, easy-to-implement, and high-throughput methodology that employs nitrocellulose membranes to immobilize lipids in order to rapid screen and identify protein-lipid interactions. In this chapter, we show how this methodology can identify potential modulators of protein-lipid interactions by screening water-soluble lipid competitors or even the introduction of pH changes during the binding assay to identify pH-dependent lipid binding events.

Analysis of Protein-Lipid Interactions Using Purified C2 Domains.

C2 domains (C2s) are regulatory protein modules identified in eukaryotic proteins targeted to cell membranes. C2s were initially characterized as independently folded Ca(2+)-dependent phospholipids binding domains; however, later studies have shown that C2s have evolutionarily diverged into Ca(2+)-dependent and Ca(2+)-independent forms. These forms interact and regulate their affinity to diverse lipid species using different binding mechanisms. In this protocol we describe a biochemical approach to produce, purify, and solubilize functional C2 domains bound to GST for the identification of their putative Ca(2+)-dependent and Ca(2+)-independent lipid-binding partners.

Analyzing Protein-Phosphoinositide Interactions with Liposome Flotation Assays.

Liposome flotation assays are a convenient tool to study protein-phosphoinositide interactions. Working with liposomes resembles physiological conditions more than protein-lipid overlay assays, which makes this method less prone to detect false positive interactions. However, liposome lipid composition must be well-considered in order to prevent nonspecific binding of the protein through electrostatic interactions with negatively charged lipids like phosphatidylserine. In this protocol we use the PROPPIN Hsv2 (homologous with swollen vacuole phenotype 2) as an example to demonstrate the influence of liposome lipid composition on binding and show how phosphoinositide binding specificities of a protein can be characterized with this method.