Routine Decontamination of Surfaces Relevant to Working Dogs: Neutralization of Superficial Coronavirus Contamination.

Given the increased deployment of working dogs to settings with pathogenic biological agents, a safe, effective, and logistically feasible surface decontamination protocol is essential to protect both the animals and their human handlers. Our group previously found that superficial contamination on surfaces relevant to the working dog community, including leashes and toys, could be significantly reduced using a standardized wiping protocol with various cleansing products. To expand upon this work, we analyzed the ability of this protocol to decontaminate surface-deposited bovine coronavirus, which was used as a BSL2 surrogate for SARS-CoV-2. Unsurprisingly, the physical characteristics of a given surface, including porosity and texture, had a significant effect on the ability to recover viable virus remaining on the surface post treatment. After correcting for these differences, however, wiping with 70% isopropyl alcohol (IPA) and 0.5% chlorhexidine performed best, reducing viral titers by >3 log on plastic bumper toys and nylon collars, and by >2 log on rubber toys and tennis balls. Leather leashes and Velcro proved more difficult to decontaminate, but both still showed significant loss of viral contamination following wiping with IPA or chlorhexidine. This work (i) validates the utility of a simple protocol for the neutralization of viruses on several surfaces, (ii) identifies materials that are more difficult to decontaminate, which should, thus, be considered for removal from field use, and (iii) highlights the need for further development of protocols testing porous or textured surfaces.

Characterization of MxiE- and H-NS-Dependent Expression of ipaH7.8, ospC1, yccE, and yfdF in Shigella flexneri.

Shigella flexneri uses a type 3 secretion system (T3SS) apparatus to inject virulence effector proteins into the host cell cytosol. Upon host cell contact, MxiE, an S. flexneri AraC-like transcriptional regulator, is required for the expression of a subset of T3SS effector genes encoded on the large virulence plasmid. Here, we defined the MxiE regulon using RNA-seq. We identified virulence plasmid- and chromosome-encoded genes that are activated in response to type 3 secretion in a MxiE-dependent manner. Bioinformatic analysis revealed that similar to previously known MxiE-dependent genes, chromosome-encoded genes yccE and yfdF contain a regulatory element known as the MxiE box, which is required for their MxiE-dependent expression. The significant AT enrichment of MxiE-dependent genes suggested the involvement of H-NS. Using a dominant negative H-NS system, we demonstrate that H-NS silences the expression of MxiE-dependent genes located on the virulence plasmid (ipaH7.8 and ospC1) and the chromosome (yccE and yfdF). Furthermore, we show that MxiE is no longer required for the expression of ipaH7.8, ospC1, yccE, and yfdF when H-NS silencing is relieved. Finally, we show that the H-NS anti-silencer VirB is not required for ipaH7.8 and yccE expression upon MxiE/IpgC overexpression. Based on these genetic studies, we propose a model of MxiE-dependent gene regulation in which MxiE counteracts H-NS-mediated silencing. IMPORTANCE The expression of horizontally acquired genes, including virulence genes, is subject to complex regulation involving xenogeneic silencing proteins, and counter-silencing mechanisms. The pathogenic properties of Shigella flexneri mainly rely on the acquisition of the type 3 secretion system (T3SS) and cognate effector proteins, whose expression is repressed by the xenogeneic silencing protein H-NS. Based on previous studies, releasing H-NS-mediated silencing mainly relies on two mechanisms involving (i) a temperature shift leading to the release of H-NS at the virF promoter, and (ii) the virulence factor VirB, which dislodges H-NS upon binding to specific motifs upstream of virulence genes, including those encoding the T3SS. In this study, we provide genetic evidence supporting the notion that, in addition to VirB, the AraC family member MxiE also contributes to releasing H-NS-mediated silencing in S. flexneri.

Design of experiments approach to developing a robust ink for bioprinting.

Despite advancements in tissue engineering, the methods used to generate three-dimensional (3D)in vitromodels for rapid screening and characterization studies remain time and labor intensive. Bioprinting offers an opportunity to offset these limitations by providing a scalable, high-throughput method with precise control over biomaterial scaffold and cellular deposition. However, the process of formulating bioinks can be complex in terms of balancing the mechanical integrity of a bioscaffold and viability of cells. One key factor, especially in alginate-based bioinks, is the rate of bioscaffold dissolution. It must allow cells to replace the bioscaffold with extracellular matrix (ECM), yet remain durable during extended tissue culture. This study uses a Design of Experiments (DoE) approach to understand the dependencies of multiple variables involved in the formulation and processing of an alginate-based bioink. The focus of the DoE was to understand the effects of hydrogel composition on bioink durability while maintaining cell viability. Three ingredients were varied in all: alginate, nanocellulose, and fibrinogen. Their effects on the bioink were then measured with respect to extrudability, strength, and stiffness as determined by dynamic mechanical analysis (DMA). The DoE demonstrated that mechanical integrity increased with increasing alginate concentration. In contrast, fibrinogen and nanofibril concentration had no statistically significant effect. The optimized ink containing fibroblasts was printable using multiple nozzle sizes while also supporting fibroblast cell viability. DMA characterization further showed that the composition of the cell culture medium did not modulate the degradation rate of the hydrogel. Ultimately, the study outlines a methodology for formulating a bioink that will result in robust bioscaffolds forin vitromodel development.

Routine Decontamination of Working Canines: A Study on the Removal of Superficial Gross Contamination.

Odor detection canines are a valuable resource used by multiple agencies for the sensitive detection of explosives, narcotics, firearms, agricultural products, and even human bodies. These canines and their handlers are frequently deployed to pathogen-contaminated environments or to work in close proximity with potentially sick individuals. Appropriate decontamination protocols must be established to mitigate both canine and handler exposure in these scenarios. Despite this potential risk, extremely limited guidance is available on routine canine decontamination from pathogenic biological materials. In this article, we evaluate the ability of several commercial off-the-shelf cleansing products, used in wipe form, to remove superficial contamination from fur, canine equipment, and toys. Using Glo Germ MIST as a proxy for biological contamination, our analysis demonstrated more than a 90% average reduction in contamination after wiping with a Nolvasan scrub solution, 0.5% chlorhexidine solution, or 70% isopropyl alcohol. Wiping with nondisinfectant baby wipes or water yielded an almost 80% average removal of contaminant from all surfaces. Additionally, researchers used Gwet's AC2 measurement to assess interrater reliability, which demonstrated substantial agreement (P < .001). These data provide key insights toward the development of a rapid, convenient, and fieldable alternative to traditional water-intensive bathing of working canines.