The Ninth Visual Object Tracking VOT2021 Challenge Results

acceptedVersionPeer reviewe

Engineering of Bacteriophage T4 Genome Using CRISPR-Cas9

Bacteriophages likely constitute the largest biomass on Earth. However, very few phage genomes have been well-characterized, the tailed phage T4 genome being one of them. Even in T4, much of the genome remained uncharacterized. The classical genetic strategies are tedious, compounded by genome modifications such as cytosine hydroxylmethylation and glucosylation which makes T4 DNA resistant to most restriction endonucleases. Here, using the type-II CRISPR-Cas9 system, we report the editing of both modified (ghm-Cytosine) and unmodified (Cytosine) T4 genomes. The modified genome, however, is less susceptible to Cas9 nuclease attack when compared to the unmodified genome. The efficiency of restriction of modified phage infection varied greatly in a spacer-dependent manner, which explains some of the previous contradictory results. We developed a genome editing strategy by codelivering into <i>E. coli</i> a CRISPR-Cas9 plasmid and a donor plasmid containing the desired mutation(s). Single and multiple point mutations, insertions and deletions were introduced into both modified and unmodified genomes. As short as 50-bp homologous flanking arms were sufficient to generate recombinants that can be selected under the pressure of CRISPR-Cas9 nuclease. A 294-bp deletion in RNA ligase gene <i>rnlB</i> produced viable plaques, demonstrating the usefulness of this editing strategy to determine the essentiality of a given gene. These results provide the first demonstration of phage T4 genome editing that might be extended to other phage genomes in nature to create useful recombinants for phage therapy applications

Design of bacteriophage T4-based artificial viral vectors for human genome remodeling

Abstract Designing artificial viral vectors (AVVs) programmed with biomolecules that can enter human cells and carry out molecular repairs will have broad applications. Here, we describe an assembly-line approach to build AVVs by engineering the well-characterized structural components of bacteriophage T4. Starting with a 120 × 86 nm capsid shell that can accommodate 171-Kbp DNA and thousands of protein copies, various combinations of biomolecules, including DNAs, proteins, RNAs, and ribonucleoproteins, are externally and internally incorporated. The nanoparticles are then coated with cationic lipid to enable efficient entry into human cells. As proof of concept, we assemble a series of AVVs designed to deliver full-length dystrophin gene or perform various molecular operations to remodel human genome, including genome editing, gene recombination, gene replacement, gene expression, and gene silencing. These large capacity, customizable, multiplex, and all-in-one phage-based AVVs represent an additional category of nanomaterial that could potentially transform gene therapies and personalized medicine

Gelsolin-Like Domain 3 Plays Vital Roles in Regulating the Activities of the Lily Villin/Gelsolin/Fragmin Superfamily.

The villin/gelsolin/fragmin superfamily is a major group of Ca2+-dependent actin-binding proteins (ABPs) involved in various cellular processes. Members of this superfamily typically possess three or six tandem gelsolin-like (G) domains, and each domain plays a distinct role in actin filament dynamics. Although the activities of most G domains have been characterized, the biochemical function of the G3 domain remains poorly understood. In this study, we carefully compared the detailed biochemical activities of ABP29 (a new member of this family that contains the G1-G2 domains of lily ABP135) and ABP135G1-G3 (which contains the G1-G3 domains of lily ABP135). In the presence of high Ca2+ levels in vitro (200 and 10 μM), ABP135G1-G3 exhibited greater actin severing and/or depolymerization and nucleating activities than ABP29, and these proteins had similar actin capping activities. However, in the presence of low levels of Ca2+ (41 nM), ABP135G1-G3 had a weaker capping activity than ABP29. In addition, ABP29 inhibited F-actin depolymerization, as shown by dilution-mediated depolymerization assay, differing from the typical superfamily proteins. In contrast, ABP135G1-G3 accelerated F-actin depolymerization. All of these results demonstrate that the G3 domain plays specific roles in regulating the activities of the lily villin/gelsolin/fragmin superfamily proteins

A Bacteriophage T4 Nanoparticle-Based Dual Vaccine against Anthrax and Plague

Following the deadly anthrax attacks of 2001, the Centers for Disease Control and Prevention (CDC) determined that Bacillus anthracis and Yersinia pestis that cause anthrax and plague, respectively, are two Tier 1 select agents that pose the greatest threat to the national security of the United States. Both cause rapid death, in 3 to 6 days, of exposed individuals. We engineered a virus nanoparticle vaccine using bacteriophage T4 by incorporating key antigens of both B. anthracis and Y. pestis into one formulation. Two doses of this vaccine provided complete protection against both inhalational anthrax and pneumonic plague in animal models. This dual anthrax-plague vaccine is a strong candidate for stockpiling against a potential bioterror attack involving either one or both of these biothreat agents. Further, our results establish the T4 nanoparticle as a novel platform to develop multivalent vaccines against pathogens of high public health significance.Bacillus anthracis and Yersinia pestis, the causative agents of anthrax and plague, respectively, are two of the deadliest pathogenic bacteria that have been used as biological warfare agents. Although Biothrax is a licensed vaccine against anthrax, no Food and Drug Administration-approved vaccine exists for plague. Here, we report the development of a dual anthrax-plague nanoparticle vaccine employing bacteriophage (phage) T4 as a platform. Using an in vitro assembly system, the 120- by 86-nm heads (capsids) of phage T4 were arrayed with anthrax and plague antigens fused to the small outer capsid protein Soc (9 kDa). The antigens included the anthrax protective antigen (PA) (83 kDa) and the mutated (mut) capsular antigen F1 and the low-calcium-response V antigen of the type 3 secretion system from Y. pestis (F1mutV) (56 kDa). These viral nanoparticles elicited robust anthrax- and plague-specific immune responses and provided complete protection against inhalational anthrax and/or pneumonic plague in three animal challenge models, namely, mice, rats, and rabbits. Protection was demonstrated even when the animals were simultaneously challenged with lethal doses of both anthrax lethal toxin and Y. pestis CO92 bacteria. Unlike the traditional subunit vaccines, the phage T4 vaccine uses a highly stable nanoparticle scaffold, provides multivalency, requires no adjuvant, and elicits broad T-helper 1 and 2 immune responses that are essential for complete clearance of bacteria during infection. Therefore, phage T4 is a unique nanoparticle platform to formulate multivalent vaccines against high-risk pathogens for national preparedness against potential bioterror attacks and emerging infections

Identification of the constructs concerning Ann5.

<p>(A) Schematic representations of the constructs concerning Ann5. (B) <i>Ann5</i>-specific primers were designed to identify the level of overexpression of the <i>Ann5</i> transcript by RT-PCR performed for 25 cycles. Total RNA was extracted from open flowers of the wild-type (WT), Lat52-GFP, Lat52-Ann5-GFP 1, 2 and 3 and Ann5Pro-Ann5 1, 2 and 3 homozygous lines. <i>EF4A</i> was used as the control. (C) Pollen grains from the WT, Lat52-GFP and Lat52-Ann5-GFP 1, 2 and 3 lines were observed by epi-fluorescence microscopy with a GFP filter. Homo, homozygous lines; Hetero, heterozygous lines. Half of the pollen grains in the heterozygous lines expressed Ann5-GFP, and the other half did not express Ann5-GFP. Bar  = 20 µm. (D) Densitometry analysis of the results presented in (C). Fluorescence intensities (arbitrary units) across the whole pollen grain were calculated. More than 50 pollen grains for each line were quantified. Values represent the means ± _SD. *P<0.05 and **P<0.01 by Student's <i>t</i> test.</p

Analysis of the cytoplasmic streaming velocity in pollen tubes of the overexpression lines.

<p>(A) The velocity of cytoplasmic streaming in pollen tubes from the overexpression lines Lat52-GFP, Lat52-Ann5-GFP 1 and 3, Lat52-G26-GFP 1 and Lat52-G257-GFP 1 under normal conditions. Pollen grains were cultured on germination medium for 4 h. Cytosolic particles exhibiting continuous movement were selected at random for velocity analysis using the Image J software. Values represent the means ± _SD (n = 25). (B) The relative velocity of cytoplasmic streaming in pollen tubes from the Lat52-GFP, Lat52-Ann5-GFP 1 and 3, Lat52-G26-GFP 1 and Lat52-G257-GFP 1 overexpression lines in response to 0.6 µM BFA. The pollen had germinated normally for 2 h, followed by 2 h in the presence of 0.6 µM BFA. The velocity of the particles of each individual line in normal conditions was normalized to 1. The relative velocity was displayed as the proportion over the control. Values represent the means ± _SD. *P<0.05 and **P<0.01 (n = 25) by Student's <i>t</i> test.</p