An Improved Workflow for the Quantification of Orthohantavirus Infection Using Automated Imaging and Flow Cytometry.

Determination of the infectious titer is a central requirement when working with pathogenic viruses. The plaque or focus assay is a commonly used but labor- and time-consuming approach for determining the infectious titer of orthohantavirus samples. We have developed an optimized virus quantification approach that relies on the fluorescence-based detection of the orthohantavirus nucleocapsid protein (N) in infected cells with high sensitivity. We present the use of flow cytometry but highlight fluorescence microscopy in combination with automated data analysis as an attractive alternative to increase the information retrieved from an infection experiment. Additionally, we offer open-source software equipped with a user-friendly graphical interface, eliminating the necessity for advanced programming skills.

Descending thoracic aortic mural ulceration is associated with postoperative spinal cord ischemia after branched endovascular aortic aneurysm repair.

OBJECTIVE: Paraplegia is one of the most feared complications after thoracoabdominal aortic aneurysm repair. The purpose of this study is to determine whether aortic thrombus characteristics are associated with spinal cord ischemia (SCI) after branched endovascular aneurysm repair (BEVAR). METHODS: From April 2011 to April 2020, 62 patients underwent elective BEVAR for thoracoabdominal aortic aneurysm and pararenal aortic aneurysms using a low-profile device and had a complete preoperative computed tomography angiography of the aorta from the sinotubular junction to the aortic bifurcation. Aortic thrombus was evaluated for thrombus thickness ≥5 mm, thrombus >2/3 of aortic circumference, and the presence of an ulcer-like thrombus. One point was assigned at each 5 mm axial image if all 3 criteria were met, resulting in a total "shaggy score" for the entire aorta. Data on demographics, procedural details, and outcomes were collected prospectively. All patients underwent a standard spinal cord protection protocol, including routine cerebrospinal fluid drainage. In July 2016, an insulin infusion protocol (IIP) was initiated to maintain postoperative blood glucose levels <120 mg/dL for 48 hours. The primary clinical end point was postoperative SCI. RESULTS: 10 (16%) patients developed postoperative SCI: 6 with transient paraparesis, 2 with persistent paraparesis, and 2 with persistent paraplegia. Patients with SCI were older, had higher shaggy scores, and were less likely to have been on an IIP. There were no significant differences in demographics, aneurysm type, or operative parameters. In a logistic multivariate regression model for SCI, age (odds ratio [OR]: 1.2 [1.1-1.4], P = .02) and shaggy score (OR: 1.2 [1.1-1.4], P = .02) were independently associated with increased risk of SCI, whereas treatment with the IIP was associated with lower risk of SCI (OR: 0.04 [0.006-0.50], P = .05). Of the individual components of the shaggy score, higher descending thoracic aortic ulcer scores were the most strongly associated with postoperative SCI (P = .009). CONCLUSIONS: Preoperative characterization of aortic wall thrombus is an important adjunctive tool for individualized clinical decision-making and patient counseling about the risk of SCI after BEVAR.

Molecular studies of rust on European aspen suggest an autochthonous relationship shaped by genotype.

Forests are at increasing risk from pathogen outbreak. Climate change for example enhance the risk of local disease outbreaks, and naturalization of exotic pathogens may follow human activities, warranting robust pest surveillance routines to support forest management. Melampsora pinitorqua (pine twisting rust) is of concern in Swedish forestry, and here we evaluate the use of visible rust scores (VRS) on its obligate summer host, European aspen (Populus tremula) as a tool for quantification of the pathogen. With use of species-specific primers, we could detect the native rust, but we failed to detect two exotic rusts (M. medusae and M. larici-populina). We found that aspen genotype determined the presence of fungal genetic markers (amplifying the ITS2 region of the fungal rDNA sequence) as well as DNA sequences specific to M. pinitorqua. We correlated VRS with the amount of fungal DNA in the same leaf, and we related the findings to aspen genotype-specific parameters such as the ability to synthesize and store leaf condensed tannins (CT). At the genotype level both positive and negative relationships were observed between CTs, fungal markers, and rust infestations. However, at the population level, foliar CT concentrations correlated negatively with general fungal- and rust-specific marker abundances. Our results, therefore, do not support the use of VRS to assess Melampsora infestation in Aspen. They do, however, suggest that the relationship between European aspen and rust infestation may be characterized as autochthonous in northern Sweden.

Advances in fluorescence microscopy for orthohantavirus research.

Orthohantaviruses are important zoonotic pathogens responsible for a considerable disease burden globally. Partly due to our incomplete understanding of orthohantavirus replication, there is currently no effective antiviral treatment available. Recently, novel microscopy techniques and cutting-edge, automated image analysis algorithms have emerged, enabling to study cellular, subcellular and even molecular processes in unprecedented detail and depth. To date, fluorescence light microscopy allows us to visualize viral and cellular components and macromolecular complexes in live cells, which in turn enables the study of specific steps of the viral replication cycle such as particle entry or protein trafficking at high temporal and spatial resolution. In this review, we highlight how fluorescence microscopy has provided new insights and improved our understanding of orthohantavirus biology. We discuss technical challenges such as studying live infected cells, give alternatives with recombinant protein expression and highlight future opportunities, for example, the application of super-resolution microscopy techniques, which has shown great potential in studies of different cellular processes and viral pathogens.

SMER28 Attenuates PI3K/mTOR Signaling by Direct Inhibition of PI3K p110 Delta.

SMER28 (Small molecule enhancer of Rapamycin 28) is an autophagy-inducing compound functioning by a hitherto unknown mechanism. Here, we confirm its autophagy-inducing effect by assessing classical autophagy-related parameters. Interestingly, we also discovered several additional effects of SMER28, including growth retardation and reduced G1 to S phase progression. Most strikingly, SMER28 treatment led to a complete arrest of receptor tyrosine kinase signaling, and, consequently, growth factor-induced cell scattering and dorsal ruffle formation. This coincided with a dramatic reduction in phosphorylation patterns of PI3K downstream effectors. Consistently, SMER28 directly inhibited PI3Kδ and to a lesser extent p110γ. The biological relevance of our observations was underscored by SMER28 interfering with InlB-mediated host cell entry of Listeria monocytogenes, which requires signaling through the prominent receptor tyrosine kinase c-Met. This effect was signaling-specific, since entry of unrelated, gram-negative Salmonella Typhimurium was not inhibited. Lastly, in B cell lymphoma cells, which predominantly depend on tonic signaling through PI3Kδ, apoptosis upon SMER28 treatment is profound in comparison to non-hematopoietic cells. This indicates SMER28 as a possible drug candidate for the treatment of diseases that derive from aberrant PI3Kδ activity.

Characterization of Hantavirus N Protein Intracellular Dynamics and Localization.

Hantaviruses are enveloped viruses that possess a tri-segmented, negative-sense RNA genome. The viral S-segment encodes the multifunctional nucleocapsid protein (N), which is involved in genome packaging, intracellular protein transport, immunoregulation, and several other crucial processes during hantavirus infection. In this study, we generated fluorescently tagged N protein constructs derived from Puumalavirus (PUUV), the dominant hantavirus species in Central, Northern, and Eastern Europe. We comprehensively characterized this protein in the rodent cell line CHO-K1, monitoring the dynamics of N protein complex formation and investigating co-localization with host proteins as well as the viral glycoproteins Gc and Gn. We observed formation of large, fibrillar PUUV N protein aggregates, rapidly coalescing from early punctate and spike-like assemblies. Moreover, we found significant spatial correlation of N with vimentin, actin, and P-bodies but not with microtubules. N constructs also co-localized with Gn and Gc albeit not as strongly as the glycoproteins associated with each other. Finally, we assessed oligomerization of N constructs, observing efficient and concentration-dependent multimerization, with complexes comprising more than 10 individual proteins.

Swinepox Virus Strains Isolated from Domestic Pigs and Wild Boar in Germany Display Altered Coding Capacity in the Terminal Genome Region Encoding for Species-Specific Genes.

Swinepox virus (SWPV) is a globally distributed swine pathogen that causes sporadic cases of an acute poxvirus infection in domesticated pigs, characterized by the development of a pathognomonic proliferative dermatitis and secondary ulcerations. More severe disease with higher levels of morbidity and mortality is observed in congenitally SWPV-infected neonatal piglets. In this study, we investigated the evolutionary origins of SWPV strains isolated from domestic pigs and wild boar. Analysis of whole genome sequences of SWPV showed that at least two different virus strains are currently circulating in Germany. These were more closely related to a previously characterized North American SWPV strain than to a more recent Indian SWPV strain and showed a variation in the SWPV-specific genome region. A single nucleotide deletion in the wild boar (wb) SWPV strain leads to the fusion of the SPV019 and SPV020 open reading frames (ORFs) and encodes a new hypothetical 113 aa protein (SPVwb020-019). In addition, the domestic pig (dp) SWPV genome contained a novel ORF downstream of SPVdp020, which encodes a new hypothetical 71aa protein (SPVdp020a). In summary, we show that SWPV strains with altered coding capacity in the SWPV specific genome region are circulating in domestic pig and wild boar populations in Germany.

Proteome and functional decline as platelets age in the circulation.

BACKGROUND: Platelets circulate in the blood of healthy individuals for approximately 7-10 days regulated by finely balanced processes of production and destruction. As platelets are anucleate we reasoned that their protein composition would change as they age and that this change would be linked to alterations in structure and function. OBJECTIVE: To isolate platelets of different ages from healthy individuals to test the hypothesis that changes in protein content cause alterations in platelet structure and function. METHODS: Platelets were separated according to thiazole orange fluorescence intensity as a surrogate indicator of mRNA content and so a marker of platelet age and then subjected to proteomics, imaging, and functional assays to produce an in-depth analysis of platelet composition and function. RESULTS: Total protein content was 45 ± 5% lower in old platelets compared to young platelets. Predictive proteomic pathway analysis identified associations with 28 biological processes, notably higher hemostasis in young platelets whilst apoptosis and senescence were higher in old platelets. Further studies confirmed platelet ageing was linked to a decrease in cytoskeletal protein and associated capability to spread and adhere, a reduction in mitochondria number, and lower calcium dynamics and granule secretion. CONCLUSIONS: Our findings demonstrate changes in protein content are linked to alterations in function as platelets age. This work delineates physical and functional changes in platelets as they age and serves as a base to examine differences associated with altered mean age of platelet populations in conditions such as immune thrombocytopenia and diabetes.

Eicosanoids in platelets and the effect of their modulation by aspirin in the cardiovascular system (and beyond).

Platelets are important players in thrombosis and haemostasis with their function being modulated by mediators in the blood and the vascular wall. Among these, eicosanoids can both stimulate and inhibit platelet reactivity. Platelet Cyclooxygenase (COX)-1-generated Thromboxane (TX)A2 is the primary prostanoid that stimulates platelet aggregation; its action is counter-balanced by prostacyclin, a product of vascular COX. Prostaglandin (PG)D2 , PGE2 and 12-hydroxyeicosatraenoic acid (HETE), or 15-HETE, are other prostanoid modulators of platelet activity, but some also play a role in carcinogenesis. Aspirin permanently inhibits platelet COX-1, underlying its anti-thrombotic and anti-cancer action. While the use of aspirin as an anti-cancer drug is increasingly encouraged, its continued use in addition to P2 Y12 receptor antagonists for the treatment of cardiovascular diseases is currently debated. Aspirin not only suppresses TXA2 but also prevents the synthesis of both known and unknown antiplatelet eicosanoid pathways, potentially lessening the efficacy of dual antiplatelet therapies. LINKED ARTICLES: This article is part of a themed section on Eicosanoids 35 years from the 1982 Nobel: where are we now? To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.8/issuetoc.