Reconstituting Spore Cortex Peptidoglycan Biosynthesis Reveals a Deacetylase That Catalyzes Transamidation.

Some bacteria survive in nutrient-poor environments and resist killing by antimicrobials by forming spores. The cortex layer of the peptidoglycan cell wall that surrounds mature spores contains a unique modification, muramic-δ-lactam, that is essential for spore germination and outgrowth. Two proteins, the amidase CwlD and the deacetylase PdaA, are required for muramic-δ-lactam synthesis in cells, but their combined ability to generate muramic-δ-lactam has not been directly demonstrated. Here we report an in vitro reconstitution of cortex peptidoglycan biosynthesis, and we show that CwlD and PdaA together are sufficient for muramic-δ-lactam formation. Our method enables characterization of the individual reaction steps, and we show for the first time that PdaA has transamidase activity, catalyzing both the deacetylation of N-acetylmuramic acid and cyclization of the product to form muramic-δ-lactam. This activity is unique among peptidoglycan deacetylases and is notable because it may involve the direct ligation of a carboxylic acid with a primary amine. Our reconstitution products are nearly identical to the cortex peptidoglycan found in spores, and we expect that they will be useful substrates for future studies of enzymes that act on the spore cortex.

Characterization of Live-Attenuated Powassan Virus Vaccine Candidates Identifies an Efficacious Prime-Boost Strategy for Mitigating Powassan Virus Disease in a Murine Model.

Powassan virus (POWV) is an emerging tick-borne virus and cause of lethal encephalitis in humans. The lack of treatment or prevention strategies for POWV disease underscores the need for an effective POWV vaccine. Here, we took two independent approaches to develop vaccine candidates. First, we recoded the POWV genome to increase the dinucleotide frequencies of CpG and UpA to potentially attenuate the virus by raising its susceptibility to host innate immune factors, such as the zinc-finger antiviral protein (ZAP). Secondly, we took advantage of the live-attenuated yellow fever virus vaccine 17D strain (YFV-17D) as a vector to express the structural genes pre-membrane (prM) and envelope (E) of POWV. The chimeric YFV-17D-POWV vaccine candidate was further attenuated for in vivo application by removing an N-linked glycosylation site within the nonstructural protein (NS)1 of YFV-17D. This live-attenuated chimeric vaccine candidate significantly protected mice from POWV disease, conferring a 70% survival rate after lethal challenge when administered in a homologous two-dose regimen. Importantly, when given in a heterologous prime-boost vaccination scheme, in which vaccination with the initial chimeric virus was followed by a protein boost with the envelope protein domain III (EDIII), 100% of the mice were protected without showing any signs of morbidity. Combinations of this live-attenuated chimeric YFV-17D-POWV vaccine candidate with an EDIII protein boost warrant further studies for the development of an effective vaccine strategy for the prevention of POWV disease.

Integrative Network Analysis of Predicted miRNA-Targets Regulating Expression of Immune Response Genes in Bovine Coronavirus Infection.

Bovine coronavirus (BCoV) infection that causes disease outbreaks among farm animals, resulting in significant economic losses particularly in the cattle industry, has the potential to become zoonotic. miRNAs, which are short non-coding segments of RNA that inhibits the expression of their target genes, have been identified as potential biomarkers and drug targets, though this potential in BCoV remains largely unknown. We hypothesize that certain miRNAs could simultaneously target multiple genes, are significantly conserved across many species, thereby demonstrating the potential to serve as diagnostic or therapeutic tools for bovine coronavirus infection. To this end, we utilized different existing and publicly available computational tools to conduct system analysis predicting important miRNAs that could affect BCoV pathogenesis. Eleven genes including CEBPD, IRF1, TLR9, SRC, and RHOA, significantly indicated in immune-related pathways, were identified to be associated with BCoV, and implicated in other coronaviruses. Of the 70 miRNAs predicted to target the identified genes, four concomitant miRNAs (bta-miR-11975, bta-miR-11976, bta-miR-22-3p, and bta-miR-2325c) were found. Examining the gene interaction network suggests IL-6, IRF1, and TP53 as key drivers. Phylogenetic analysis revealed that miR-22 was completely conserved across all 14 species it was searched against, suggesting a shared and important functional role. Functional annotation and associated pathways of target genes, such as positive regulation of cytokine production, IL-6 signaling pathway, and regulation of leukocyte differentiation, indicate the miRNAs are major participants in multiple aspects of both innate and adaptive immune response. Examination of variants evinced a potentially deleterious SNP in bta-miR-22-3p and an advantageous SNP in bta-miR-2325c. Conclusively, this study provides new insight into miRNAs regulating genes responding to BCoV infection, with bta-miR-22-3p particularly indicated as a potential drug target or diagnostic marker for bovine coronavirus.

Dissecting Transcription Factor-Target Interaction in Bovine Coronavirus Infection.

Coronaviruses are RNA viruses that cause significant disease within many species, including cattle. Bovine coronavirus (BCoV) infects cattle and wild ruminants, both as a respiratory and enteric pathogen, and possesses a significant economic threat to the cattle industry. Transcription factors are proteins that activate or inhibit transcription through DNA binding and have become new targets for disease therapies. This study utilized in silico tools to identify potential transcription factors that can serve as biomarkers for regulation of BCoV pathogenesis in cattle, both for testing and treatment. A total of 11 genes were identified as significantly expressed during BCoV infection through literature searches and functional analyses. Eleven transcription factors were predicted to target those genes (AREB6, YY1, LMO2, C-Rel, NKX2-5, E47, RORAlpha1, HLF, E4BP4, ARNT, CREB). Function, network, and phylogenetic analyses established the significance of many transcription factors within the immune response. This study establishes new information on the transcription factors and genes related to host-pathogen interactome in BCoV infection, particularly transcription factors YY1, AREB6, LMO2, and NKX2, which appear to have strong potential as diagnostic markers, and YY1 as a potential target for drug therapies.

Evaluation of a Therapeutic Interchange from Fluticasone/Salmeterol to Mometasone/Formoterol in Patients with Chronic Obstructive Pulmonary Disease.

BACKGROUND: Combination treatment with an inhaled corticosteroid and long-acting beta2-agonist is among the many treatment options for chronic obstructive pulmonary disease (COPD) that has been shown to improve clinical outcomes. While mometasone/formoterol does not currently have an FDA-approved indication for COPD, evidence from 2 phase 3 trials demonstrated that mometasone/formoterol can improve lung function and was well tolerated in patients with moderate-to-very severe COPD. Based on these data, a therapeutic interchange was implemented in the Kaiser Permanente Mid-Atlantic States region to convert patients with a COPD diagnosis from fluticasone/salmeterol to mometasone/formoterol. OBJECTIVE: To evaluate the impact of a therapeutic interchange from fluticasone/salmeterol to mometasone/formoterol on health outcomes in patients with COPD in a large ambulatory and managed care setting. METHODS: The investigators retrospectively reviewed the electronic medical records of patients with a COPD diagnosis who had a prescription for fluticasone/salmeterol converted to mometasone/formoterol between March 6, 2011, to March 6, 2013. Kaiser Permanente's Pharmacy and Therapeutics Committee provided recommended equivalent doses for conversion from fluticasone/salmeterol to mometasone/formoterol. Nonetheless, the final approval for the change in medication and selection of the dose was left to each physician's clinical judgment. Patients were excluded if they were (a) prescribed fluticasone/salmeterol 100/50 mcg, which has no equivalent mometasone/formoterol dose; (b) less than aged 18 years; or (c) prescribed fluticasone/salmeterol for a duration of less than 6 months preconversion to mometasone/formoterol. In addition, patients who left the Kaiser Permanente network or became deceased during the study period of interest were excluded. After the application of the inclusion and exclusion criteria, 521 patients were included in the data analysis. The primary endpoint was the determination of the difference in the occurrence of COPD exacerbations 6 months pre- and postconversion from fluticasone/salmeterol to mometasone/formoterol. COPD exacerbations were defined by the diagnosis or documentation of a COPD exacerbation during any hospitalizations, urgent care (UC)/emergency department (ED) visits, or clinic encounters. Secondary outcomes included the determination of the difference in the occurrence of intensive care unit admissions, hospitalizations, UC/ED visits, and clinic encounters for COPD exacerbations 6 months pre- and postconversion; number of patients who required modification in therapy; and any reasons for mometasone/for-moterol discontinuation postconversion. Patients served as their own controls to compare any differences in outcomes while taking mometasone/formoterol versus fluticasone/salmeterol. RESULTS: Within our patient population, 34.2% (n = 178) of patients experienced at least 1 COPD exacerbation while prescribed fluticasone/salmeterol compared with 28.6% (n = 149) of patients while prescribed mometasone/formoterol (P = 0.030). Mometasone/formoterol therapy did not demonstrate any statistically significant differences in the secondary outcomes (P < 0.050). A later subgroup analysis of the primary outcome revealed that factors associated with a statistically significant decrease in the occurrence of COPD exacerbations were male sex (P = 0.023), comorbid asthma (P = 0.026), and conversion from fluticasone/salmeterol to a more potent dose of mometasone/formoterol (P = 0.014). CONCLUSIONS: There was a statistically significant decrease in the proportion of patients who experienced COPD exacerbations postconversion from fluticasone/salmeterol to mometasone/formoterol. This study is an example of a real-world therapeutic interchange that provides additional data to support the use of mometasone/formoterol for its unlabeled COPD indication. DISCLOSURES: No outside funding supported this study. The authors report no financial or other conflicts of interest related to the subject of this article. All authors contributed to study design and manuscript revision. Yip collected and analyzed data and prepared the manuscript.

Pre-treatment choroidal thickness is not predictive of susceptibility to form-deprivation myopia in chickens.

PURPOSE: Chicks developing experimentally-induced myopia show profound thinning of the choroid. We observed a wide range of choroidal thicknesses in a sample of normal chicks prior to their use in a pedigree-based study of form-deprivation myopia. Hence, we tested whether pre-treatment choroidal thickness predicted susceptibility to myopia. METHODS: Retinal, choroidal and scleral thickness were measured using A-scan ultrasonography in normal White Leghorn chicks (n= 891) aged 4 days old, and again (n=498) after 4 days of monocular form-deprivation at age 8 days of age. Refractive error was assessed by retinoscopy. Relationships between pre-treatment choroidal thickness and other variables were investigated using general linear models and variance components analysis. RESULTS: Untreated 4 day-old male chicks had choroids approximately 10% thinner than females (p<0.001), but sex explained <2% of the overall variability in choroidal thickness. Axial eye length in these untreated chicks was not significantly associated with choroidal thickness (p=0.25). Moreover, pre-treatment choroidal thickness was not predictive of susceptibility to form-deprivation myopia (p=0.89). Heritability analysis suggested that at least 50% of the variation in pre-treatment choroidal thickness was determined by additive genetic effects (p<0.001). CONCLUSIONS: Parental choroidal thickness is the major determinant of choroidal thickness in untreated 4-day old chicks. Despite choroidal thickness potentially being indicative of ongoing emmetropisation to innate refractive errors, in this study it was not predictive of subsequent susceptibility to form-deprivation myopia.