Necrotizing plasma cell-rich aortitis and sudden cardiac death: Late sequelae of COVID-19?

The ongoing epidemic caused by the coronavirus SARS-CoV-2 is characterized by a variety of pathologic processes within the syndrome of COVID-19. Usually beginning as an upper respiratory infection with potential progression to a pneumonitis, many cases of COVID-19 that show minimal signs or symptoms initially may develop adverse systemic sequelae later, such as widespread thrombo-embolic phenomena, systemic inflammatory disorders (especially in children), or vasculitis. Here, we present a patient who suffered a sudden cardiac death following persistent SARS-CoV-2 viral positivity for four-and-one-half months after a mild clinical viral course. At routine autopsy, a remarkable plasma cell-rich necrotizing aortitis was uncovered. The aortic intima displayed diffuse, circumferential ongoing chronic intimal edema, inflammation, and neo-vascularization. The plasma cell-rich inflammatory process also involved the origin of the left main coronary artery (LM) causing a coronary arteritis accompanied by subacute, stenosing intimal vascular smooth muscle cell (VSMC) proliferation resulting in acute myocardial necrosis as a cause of death. A similar vasculitis and plaque were noted during the routine autopsy at the ostium of the celiac artery; vasculitis was not found systemically or in smaller caliber vessels. Through a variety of techniques including extensive histopathologic and immunohistochemical characterization, immunostaining localization of viral antigen, and transmission electron microscopy we present highly suggestive evidence that this unique necrotizing, plasma cell-rich aortitis is a rare sequela of COVID-19.

Exosomally Targeting microRNA23a Ameliorates Microvascular Endothelial Barrier Dysfunction Following Rickettsial Infection.

Spotted fever group rickettsioses caused by Rickettsia (R) are devastating human infections, which mainly target microvascular endothelial cells (ECs) and can induce lethal EC barrier dysfunction in the brain and lungs. Our previous evidence reveals that exosomes (Exos) derived from rickettsial-infected ECs, namely R-ECExos, can induce disruption of the tight junctional (TJ) protein ZO-1 and barrier dysfunction of human normal recipient brain microvascular endothelial cells (BMECs). However, the underlying mechanism remains elusive. Given that we have observed that microRNA23a (miR23a), a negative regulator of endothelial ZO-1 mRNA, is selectively sorted into R-ECExos, the aim of the present study was to characterize the potential functional role of exosomal miR23a delivered by R-ECExos in normal recipient BMECs. We demonstrated that EC-derived Exos (ECExos) have the capacity to deliver oligonucleotide RNAs to normal recipient BMECs in an RNase-abundant environment. miR23a in ECExos impairs normal recipient BMEC barrier function, directly targeting TJ protein ZO-1 mRNAs. In separate studies using a traditional in vitro model and a novel single living-cell biomechanical assay, our group demonstrated that miR23a anti-sense oligonucleotide-enriched ECExos ameliorate R-ECExo-provoked recipient BMEC dysfunction in association with stabilization of ZO-1 in a dose-dependent manner. These results suggest that Exo-based therapy could potentially prove to be a promising strategy to improve vascular barrier function during bacterial infection and concomitant inflammation.

Cell radiolabeling with acoustophoresis cell washing.

Labeling immune cells with zirconium-89 (89Zr)-oxine has become a viable method to track cells in vivo by PET in various pre-clinical animal models and in clinical applications. Currently, 89Zr-oxine cell labeling is performed manually, which requires a highly trained specialist and is prone to human error. As the first phase in developing a fully automated radiolabeling system to address this problem, we assess the use of acoustophoresis cell washing to replace the centrifugal cell washing used in the current 89Zr-oxine cell radiolabeling procedure. To accomplish this, a cell radiolabeling procedure was developed in which two steps requiring a centrifuge to wash cells were replaced using acoustophoresis cell washing methods. The process was tested using murine EL4 lymphoma and T cells. The centrifuge cell labeling procedure was used as a control to compare the acoustophoresis cell washing procedure. The acoustophoresis method produced radiolabeled cells with similar properties to the centrifugal method when comparing labeling efficiency, labeled specific activity, efficacy of removing unbound 89Zr-oxine from the suspension, cell viability measured using annexin V/propidium iodide staining and activation function. This suggests that acoustophoresis cell washing can be used in the design of an automated benchtop, good manufacture practice-qualified acoustophoresis cell radiolabeling device.

Host EPAC1 Modulates Rickettsial Adhesion to Vascular Endothelial Cells via Regulation of ANXA2 Y23 Phosphorylation.

INTRODUCTION: Intracellular cAMP receptor exchange proteins directly activated by cAMP 1 (EPAC1) regulate obligate intracellular parasitic bacterium rickettsial adherence to and invasion into vascular endothelial cells (ECs). However, underlying precise mechanism(s) remain unclear. The aim of the study is to dissect the functional role of the EPAC1-ANXA2 signaling pathway during initial adhesion of rickettsiae to EC surfaces. METHODS: In the present study, an established system that is anatomically based and quantifies bacterial adhesion to ECs in vivo was combined with novel fluidic force microscopy (FluidFM) to dissect the functional role of the EPAC1-ANXA2 signaling pathway in rickettsiae-EC adhesion. RESULTS: The deletion of the EPAC1 gene impedes rickettsial binding to endothelium in vivo. Rickettsial OmpB shows a host EPAC1-dependent binding strength on the surface of a living brain microvascular EC (BMEC). Furthermore, ectopic expression of phosphodefective and phosphomimic mutants replacing tyrosine (Y) 23 of ANXA2 in ANXA2-knock out BMECs results in different binding force to reOmpB in response to the activation of EPAC1. CONCLUSIONS: EPAC1 modulates rickettsial adhesion, in association with Y23 phosphorylation of the binding receptor ANXA2. Underlying mechanism(s) should be further explored to delineate the accurate role of cAMP-EPAC system during rickettsial infection.

Pathogenomes of Atypical Non-shigatoxigenic Escherichia coli NSF/SF O157:H7/NM: Comprehensive Phylogenomic Analysis Using Closed Genomes.

The toxigenic conversion of Escherichia coli strains by Shiga toxin-converting (Stx) bacteriophages were prominent and recurring events in the stepwise evolution of enterohemorrhagic E. coli (EHEC) O157:H7 from an enteropathogenic (EPEC) O55:H7 ancestor. Atypical, attenuated isolates have been described for both non-sorbitol fermenting (NSF) O157:H7 and SF O157:NM serotypes, which are distinguished by the absence of Stx, the characteristic virulence hallmark of Stx-producing E. coli (STEC). Such atypical isolates either never acquired Stx-phages or may have secondarily lost stx during the course of infection, isolation, or routine subculture; the latter are commonly referred to as LST (Lost Shiga Toxin)-isolates. In this study we analyzed the genomes of 15 NSF O157:H7 and SF O157:NM strains from North America, Europe, and Asia that are characterized by the absence of stx, the virulence hallmark of STEC. The individual genomic basis of the Stx (-) phenotype has remained largely undetermined as the majority of STEC genomes in public genome repositories were generated using short read technology and are in draft stage, posing a major obstacle for the high-resolution whole genome sequence typing (WGST). The application of LRT (long-read technology) sequencing provided us with closed genomes, which proved critical to put the atypical non-shigatoxigenic NSF O157:H7 and SF O157:NM strains into the phylogenomic context of the stepwise evolutionary model. Availability of closed chromosomes for representative Stx (-) NSF O157:H7 and SF O157:NM strains allowed to describe the genomic basis and individual evolutionary trajectories underlying the absence of Stx at high accuracy and resolution. The ability of LRT to recover and accurately assemble plasmids revealed a strong correlation between the strains' featured plasmid genotype and chromosomally inferred clade, which suggests the coevolution of the chromosome and accessory plasmids. The identified ancestral traits in the pSFO157 plasmid of NSF O157:H7 strain LSU-61 provided additional evidence for its intermediate status. Taken together, these observations highlight the utility of LRTs for advancing our understanding of EHEC O157:H7/NM pathogenome evolution. Insights into the genomic and phenotypic plasticity of STEC on a lineage- and genome-wide scale are foundational to improve and inform risk assessment, biosurveillance, and prevention strategies.

Two new phytoecdysteroids from Sphenocentrum jollyanum Pierre root.

The crude methanol extract of Sphenocentrum jollyanum root exhibited 98% and 80% antimicrobial activity against Aspergillus fumigatus Pinh and Vancomycin resistant enterococcus (VRE) at a concentration of 200 µg/mL, with IC50 11.45 and 12.95 µg/mL, respectively. The ethyl acetate fraction of methanol extract showed in-vitro antimicrobial activity against A. fumigatus Pinh at 83% with IC50 of <8 µg/mL. The phytochemical investigation of ethyl acetate fraction yielded six compounds, which were identified by their NMR, IR and MS spectral analyses as two new phytoecdysteroidal glycosides Sphenocentroside A (1), and Sphenocentroside B (2), and four known phytoecdysteroids: polypodoaurein (3), polypodine B (4), ecdysterone (5), and 20, 26-dihydroxyecdysone (6).

Closed Genome Sequence of Escherichia coli K-12 Group Strain C600.

Escherichia coli strain C600 is a prototypical K-12 derived laboratory strain which has been broadly used for molecular microbiology and bacterial physiology studies since its isolation in 1954. Here, we present the closed genome sequence of E. coli strain C600, retrieved from the American Type Culture Collection (ATCC 23724).

In vitro and in vivo antimicrobial evaluation of alkaloidal extracts of Enantia chlorantha stem bark and their formulated ointments.

The in vitro and in vivo antimicrobial evaluation of the formulated ointment of alkaloidal extract of Enantia chlorantha Oliv. (Annonaceae) was the concern of this study. The alkaloidal fraction of the stem bark extract was formulated into simple ointment using British Pharmacopoeia formula for preparation of simple ointment. Agar diffusion and agar dilution methods were used for the in vitro antimicrobial studies. Ketoconazole 4000 µg/mL and tioconazole cream 1% were used as reference standards while normal saline was used as control. The fungicidal activity kinetics of the plant extract was carried out using selected concentrations of the plant extract against the most sensitive organism (Candida albicans). For the in vivo studies, 25 albino rats weighing between 180-200 g were divided into 5 groups, anesthesized (thiopental sodium 50 mg/kg), infected with overnight culture of Candida albicans and incubated at 37 degrees C for three days to allow for growth of the microorganisms. Each of the five groups was treated on the third day of incubation with different concentrations of the formulated simple ointment (200 mg/mL, 100 mg/kg, 50 mg/mL), tioconazole cream 1% (reference standard) and normal saline control, respectively. The alkaloidal extract exhibited greater zones of inhibition with Candida glabrata and Trichophyton tonsurans while Candida albicans and Trichophyton interdigitali also showed some sensitivity. There was no surviving organism at the end of 240 min at 100 mg/mL concentration with 10(-4) dilution factor. Treatment of the infected rats with the formulated simple ointments (200, 100 and 50 mg/mL) showed that 50 mg/mL ointment had a better percentage reduction in the fungal loads at the end of the experiment when compared with the 200 mg/mL simple ointment as well as the standard tioconazole 1% cream and normal saline treated rats, respectively. The alkaloidal fraction of Enantia chlorantha stem bark as well as the formulated ointment exhibited significant in vitro and in vivo antifungal activities against different species of Candida, dermatophytes and plant fungi.