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1. The Effects of Packaged, but Misguided, Single-Stranded DNA Genomes Are Transmitted to the Outer Surface of the ϕX174 Capsid

   https://doi.org/10.1128/JVI.00883-21  

   Ogunbunmi, Elizabeth T.; Roznowski, Aaron P.; Fane, Bentley A. (August 2021, Journal of Virology)

   Sandri-Goldin, Rozanne M. (Ed.)

   ABSTRACT Most icosahedral viruses condense their genomes into volumetrically constrained capsids. However, concurrent genome biosynthesis and packaging are specific to single-stranded DNA (ssDNA) viruses. ssDNA genome packaging combines elements found in both double-stranded DNA (dsDNA) and ssRNA systems. Similar to dsDNA viruses, the genome is packaged into a preformed capsid. Like ssRNA viruses, there are numerous capsid-genome associations. In ssDNA microviruses, the DNA-binding protein J guides the genome between 60 icosahedrally ordered DNA binding pockets. It also partially neutralizes the DNA’s negative phosphate backbone. ϕX174-related microviruses, such as G4 and α3, have J proteins that differ in length and charge organization. This suggests that interchanging J proteins could alter the path used to guide DNA in the capsid. Previously, a ϕXG4J chimera, in which the ϕX174 J gene was replaced with the G4 gene, was characterized. It displayed lethal packaging defects, which resulted in procapsids being removed from productive assembly. Here, we report the characterization of another inviable chimera, ϕXα3J. Unlike ϕXG4J, ϕXα3J efficiently packaged DNA but produced noninfectious particles. These particles displayed a reduced ability to attach to host cells, suggesting that internal DNA organization could distort the capsid’s outer surface. Mutations that restored viability altered J-coat protein contact sites. These results provide evidence that the organization of ssDNA can affect both packaging and postpackaging phenomena. IMPORTANCE ssDNA viruses utilize icosahedrally ordered protein-nucleic acids interactions to guide and organize their genomes into preformed shells. As previously demonstrated, chaotic genome-capsid associations can inhibit ϕX174 packaging by destabilizing packaging complexes. However, the consequences of poorly organized genomes may extend beyond the packaging reaction. As demonstrated herein, it can lead to uninfectious packaged particles. Thus, ssDNA genomes should be considered an integral and structural virion component, affecting the properties of the entire particle, which includes the capsid’s outer surface. 

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2. Effects of Pseudomonas aeruginosa on Microglial-Derived Extracellular Vesicle Biogenesis and Composition

   https://doi.org/10.3390/pathogens8040297  

   Jones, Leandra B.; Kumar, Sanjay; Bell, Courtnee’ R.; Peoples, Veolonda A.; Crenshaw, Brennetta J.; Coats, Mamie T.; Scoffield, Jessica A.; Rowe, Glenn C.; Sims, Brian; Matthews, Qiana L. (December 2019, Pathogens)

   The packaging of molecular constituents inside extracellular vesicles (EVs) allows them to participate in intercellular communication and the transfer of biological molecules, however the role of EVs during bacterial infection is poorly understood. The goal of this study was to examine the effects of Pseudomonas aeruginosa (P. aeruginosa) infection on the biogenesis and composition of EVs derived from the mouse microglia cell line, BV-2. BV-2 cells were cultured in exosome-free media and infected with 0, 1.3 × 104, or 2.6 × 104 colony forming units per milliliter P. aeruginosa for 72 h. The results indicated that compared with the control group, BV-2 cell viability significantly decreased after P. aeruginosa infection and BV-2-derived EVs concentration decreased significantly in the P. aeruginosa-infected group. P. aeruginosa infection significantly decreased chemokine ligand 4 messenger RNA in BV-2-derived infected EVs, compared with the control group (p ≤ 0.05). This study also revealed that heat shock protein 70 (p ≤ 0.05) and heat shock protein 90β (p ≤ 0.001) levels of expression within EVs increased after P. aeruginosa infection. EV treatment with EVs derived from P. aeruginosa infection reduced cell viability of BV-2 cells. P. aeruginosa infection alters the expression of specific proteins and mRNA in EVs. Our study suggests that P. aeruginosa infection modulates EV biogenesis and composition, which may influence bacterial pathogenesis and infection. 

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3. Alcohol Exposure Impacts the Composition of HeLa-Derived Extracellular Vesicles

   https://doi.org/10.3390/biomedicines7040078  

   Jones, Leandra B.; Kumar, Sanjay; Curry, Aliyah J.; Price, Jayde S.; Krendelchtchikov, Alexandre; Crenshaw, Brennetta J.; Bell, Courtnee’ R.; Williams, Sparkle D.; Tolliver, Tambre A.; Saldanha, Sabita N.; et al (December 2019, Biomedicines)

   Extracellular vesicles are nanosized vesicles that are under intense investigation for their role in intercellular communication. Extracellular vesicles have begun to be examined for their role in disease protection and their role as disease biomarkers and/or vaccine agents. The goal of this study was to investigate the effects of alcohol exposure on the biogenesis and composition of extracellular vesicles derived from the cervical cancer line, HeLa. The HeLa cells were cultured in exosome-free media and were either mock-treated (control) or treated with 50 mM or 100 mM of alcohol for 24 h and 48 h. Our results demonstrated that alcohol significantly impacts HeLa cell viability and exosome biogenesis/composition. Importantly, our studies demonstrate the critical role of alcohol on HeLa cells, as well as HeLa-derived extracellular vesicle biogenesis and composition. Specifically, these results indicate that alcohol alters extracellular vesicles’ packaging of heat shock proteins and apoptotic proteins. Extracellular vesicles serve as communicators for HeLa cells, as well as biomarkers for the initiation and progression of disease. 

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4. Deep learning provides high accuracy in automated chondrocyte viability assessment in articular cartilage using nonlinear optical microscopy

   https://doi.org/10.1364/BOE.417478  

   Chen, Xun; Li, Yang; Wyman, Nicole; Zhang, Zheng; Fan, Hongming; Le, Michael; Gannon, Steven; Rose, Chelsea; Zhang, Zhao; Mercuri, Jeremy; et al (April 2021, Biomedical Optics Express)

   Chondrocyte viability is a crucial factor in evaluating cartilage health. Most cell viability assays rely on dyes and are not applicable forin vivoor longitudinal studies. We previously demonstrated that two-photon excited autofluorescence and second harmonic generation microscopy provided high-resolution images of cells and collagen structure; those images allowed us to distinguish live from dead chondrocytes by visual assessment or by the normalized autofluorescence ratio. However, both methods require human involvement and have low throughputs. Methods for automated cell-based image processing can improve throughput. Conventional image processing algorithms do not perform well on autofluorescence images acquired by nonlinear microscopes due to low image contrast. In this study, we compared conventional, machine learning, and deep learning methods in chondrocyte segmentation and classification. We demonstrated that deep learning significantly improved the outcome of the chondrocyte segmentation and classification. With appropriate training, the deep learning method can achieve 90% accuracy in chondrocyte viability measurement. The significance of this work is that automated imaging analysis is possible and should not become a major hurdle for the use of nonlinear optical imaging methods in biological or clinical studies. 

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5. Cryopreservation of 3D Bioprinted Scaffolds with Temperature-Controlled-Cryoprinting

   https://doi.org/10.3390/gels9060502  

   Warburton, Linnea; Rubinsky, Boris (June 2023, Gels)

   Temperature-Controlled-Cryoprinting (TCC) is a new 3D bioprinting technology that allows for the fabrication and cryopreservation of complex and large cell-laden scaffolds. During TCC, bioink is deposited on a freezing plate that descends further into a cooling bath, keeping the temperature at the nozzle constant. To demonstrate the effectiveness of TCC, we used it to fabricate and cryopreserve cell-laden 3D alginate-based scaffolds with high cell viability and no size limitations. Our results show that Vero cells in a 3D TCC bioprinted scaffold can survive cryopreservation with a viability of 71%, and cell viability does not decrease as higher layers are printed. In contrast, previous methods had either low cell viability or decreasing efficacy for tall or thick scaffolds. We used an optimal temperature profile for freezing during 3D printing using the two-step interrupted cryopreservation method and evaluated drops in cell viability during the various stages of TCC. Our findings suggest that TCC has significant potential for advancing 3D cell culture and tissue engineering. 

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