Differential pathogenesis of closely related 2018 Nigerian outbreak clade III Lassa virus isolates

Nigeria continues to experience ever increasing annual outbreaks of Lassa fever (LF). The World Health Organization has recently declared Lassa virus (LASV) as a priority pathogen for accelerated research leading to a renewed international effort to develop relevant animal models of disease and effective countermeasures to reduce LF morbidity and mortality in endemic West African countries. A limiting factor in evaluating medical countermeasures against LF is a lack of well characterized animal models outside of those based on infection with LASV strain Josiah originating form Sierra Leone, circa 1976. Here we genetically characterize five recent LASV isolates collected from the 2018 outbreak in Nigeria. Three isolates were further evaluated in vivo and despite being closely related and from the same spatial / geographic region of Nigeria, only one of the three isolates proved lethal in strain 13 guinea pigs and non-human primates (NHP). Additionally, this isolate exhibited atypical pathogenesis characteristics in the NHP model, most notably respiratory failure, not commonly described in hemorrhagic cases of LF. These results suggest that there is considerable phenotypic heterogeneity in LASV infections in Nigeria, which leads to a multitude of pathogenesis characteristics that could account for differences between subclinical and lethal LF infections. Most importantly, the development of disease models using currently circulating LASV strains in West Africa are critical for the evaluation of potential vaccines and medical countermeasures.     

Artifactual FA dimers mimic FAHFA signals in untargeted metabolomics pipelines

FA esters of hydroxy FAs (FAHFAs) are lipokines with extensive structural and regional isomeric diversity that impact multiple physiological functions, including insulin sensitivity and glucose homeostasis. Because of their low molar abundance, FAHFAs are typically quantified using highly sensitive LC-MS/MS methods. Numerous relevant MS databases house in silico-spectra that allow identification and speciation of FAHFAs. These provisional chemical feature assignments provide a useful starting point but could lead to misidentification. To address this possibility, we analyzed human serum with a commonly applied high-resolution LC-MS untargeted metabolomics platform. We found that many chemical features are putatively assigned to the FAHFA lipid class based on exact mass and fragmentation patterns matching spectral databases. Careful validation using authentic standards revealed that many investigated signals provisionally assigned as FAHFAs are in fact FA dimers formed in the LC-MS pipeline. These isobaric FA dimers differ structurally only by the presence of an olefinic bond. Furthermore, stable isotope-labeled oleic acid spiked into human serum at subphysiological concentrations showed concentration-dependent formation of a diverse repertoire of FA dimers that analytically mimicked FAHFAs. Conversely, validated FAHFA species did not form spontaneously in the LC-MS pipeline. Together, these findings underscore that FAHFAs are endogenous lipid species.  However, nonbiological FA dimers forming in the setting of high concentrations of FFAs can be misidentified as FAHFAs. Based on these results, we assembled a FA dimer database to identify nonbiological FA dimers in untargeted metabolomics datasets.     

Cloning and characterization of a region responsible for the maintenance of megaplasmid pTAV3 of Paracoccus versutus UW1

Using cointegrate formation, we constructed a basic replicon of the megaplasmid/mini-chromosome pTAV3 of Paracoccus versutus UW1. It is composed of two adjacent modules, responsible for plasmid replication (rep) and partitioning (par). Functional analysis of the par region identified a determinant of incompatibility (inc2), whose presence is crucial for proper partitioning (the partitioning site). Database searches revealed that the only known replicon with significant homology to that of pTAV3 is encoded by the chromosome cII of Rhodobacter sphaeroides 2.4.1. Incompatibility studies showed that closely related basic replicons are also encoded by megaplasmids (above 400 kb) harbored by four strains of P. pantotrophus. Basic replicons of the pTAV3-type are able to maintain large bacterial genomes, therefore they appear to be good candidates for the construction of vectors specific for Alphaproteobacteria.     

Preincubation with sodium ascorbate potentiates insulin-dependent PKB/Akt and c-Jun phosphorylation in L6 rat myoblasts challenged with reactive oxygen/nitrogen species

Previously, we reported that mitogenicity in L6 muscle cells was stimulated by insulin but inhibited by reactive oxygen/nitrogen species (ROS/RNS; []) and that preincubation with sodium ascorbate (ASC) protected from either the impaired DNA synthesis and/or loss of cell viability. Now, we addressed the question how ascorbate (AA) rescued DNA synthesis in L6 muscle cells being challenged with ROS/RNS. We assumed that AA might be able to influence insulin signaling. We found that insulin elevated the protein levels of both PKB/Akt kinase phosphorylated at Serine(473) (pS473-Akt), and c-Jun phosphorylated at Serine63, Serine73 (pS63, pS73-c-Jun) residues, respectively. A short-term treatment experiment (0 - 45 min) revealed that either insulin (0.1 muM) or hydrogen peroxide (0.1, 0.5 mM; H2O2) increased the pS473-Akt and pS63, pS73-c-Jun protein levels, although the effect of ROS/RNS peaked earlier (5 min) than that of insulin (45 min). Astonishingly, the elevated levels of both pS473-Akt and pS63, pS73-c-Jun in response to insulin were reduced by the concomitant treatment with H2O2 in a dose-dependent fashion. In contrast, a 4-hour preincubation with ASC (1 mM) augmented the signal from pS473-Akt and pS63, pS73-c-Jun, when both insulin and H2O2 were added. Moreover, a 24 h preincubation with ASC also elevated the pS473-Akt and pS63, pS73-c-Jun levels in response to insulin irrespective to ROS/RNS co-treatment. During chronic treatment studies, ROS/RNS stimulated neither phosphorylation of Akt nor c-Jun, indicating that ROS/RNS-dependent activation of the above-mentioned proteins was short-term and transient. Furthermore, higher levels of pS473 Akt and pS63, pS73-c-Jun after preincubation with ASC suggest that by this route AA could protect insulin-induced mitogenicity. Basal levels of Akt and its target p70(S6K) remained constant regardless of treatment. These results suggest that AA defends the insulin-stimulated mitogenicity hampered by ROS/RNS most likely by the amplification of insulin signal at the level of pS473-Akt and pS63, pS73-c-Jun, respectively.     

Variation in melanin pigmentation of a sexually selected plumage trait and its adaptive value in the Common Snipe Gallinago gallinago

There is increasing evidence that melanin‐based plumage coloration correlates with different components of fitness and that it may act as a social or sexual signal of individual quality. We analysed variation in melanin pigmentation in the outermost tail feathers of the Common Snipe Gallinago gallinago. During courtship flights, male Snipe use their outermost tail feathers to generate a drumming sound, which plays a role in territory establishment and mate choice. As the outermost tail feathers are displayed to females during these flights, we predicted that conspicuous variation in their rusty‐brown (pheomelanin‐based) coloration may act as an honest signal of individual quality. To test this prediction, we spectrophotometrically measured brightness (an indicator of total melanin content) and red chroma (an indicator of pheomelanin content) of the outermost tail feathers in 180 juvenile and adult Common Snipe. An age‐related decline in feather brightness was found exclusively in females, suggesting that melanization could have evolved by natural selection to camouflage incubating birds. In both sexes, brightness of the tail feathers was inversely correlated with their structural quality (as measured with mass–length residuals), suggesting that melanization could increase mechanical properties of feathers and, in males, enhance the quality of courtship sonation. Red chroma positively correlated with total plasma protein concentration, supporting our prediction that pheomelanin pigmentation of tail feathers may act as an honest signal of condition. Our study indicated that variation in the melanin‐based coloration of the outermost tail feathers in the Common Snipe could have evolved as a result of several different selection pressures and it emphasizes the complexity of the processes that underlie the evolution of melanin‐based plumage coloration in birds.     

Increased yield and selected properties of bacterial cellulose exposed to different modes of a rotating magnetic field

Rotating magnetic field (RMF) is an interesting alternative to conventional bacterial cellulose (BC) production methods. The BC synthesis processes may be affected by RMF, which facilitates the transfer of oxygen and nutrients from the media to the microbial cells. RMF may also directly influence the various physical and chemical properties of BC. The main aim of the present study was to evaluate the impact of the RMF on the BC in regard to its yield and material properties. The correlation between the efficiency of polymer production and the different time of exposure to the RMF was also analyzed to determine the conditions of lower energy consumption during the cellulose formation process. It was found that the Gluconacetobacter xylinus cultures exposed to the RMF for a half of the time of the entire cellulose production process (72 h), considering the results obtained in controls, synthesized BC more effectively than bacteria continuously exposed to the RMF for 144 h. Furthermore, the application of the RMF, regardless of the exposure mode, did not negatively affect the polymer material properties. It was concluded that the use of the RMF may provide a novel technique for altering cellulose biogenesis and may be used in multiple biotechnological applications.     

RUNX1B Expression Is Highly Heterogeneous and Distinguishes Megakaryocytic and Erythroid Lineage Fate in Adult Mouse Hematopoiesis

The Core Binding Factor (CBF) protein RUNX1 is a master regulator of definitive hematopoiesis, crucial for hematopoietic stem cell (HSC) emergence during ontogeny. RUNX1 also plays vital roles in adult mice, in regulating the correct specification of numerous blood lineages. Akin to the other mammalian Runx genes, Runx1 has two promoters P1 (distal) and P2 (proximal) which generate distinct protein isoforms. The activities and specific relevance of these two promoters in adult hematopoiesis remain to be fully elucidated. Utilizing a dual reporter mouse model we demonstrate that the distal P1 promoter is broadly active in adult hematopoietic stem and progenitor cell (HSPC) populations. By contrast the activity of the proximal P2 promoter is more restricted and its upregulation, in both the immature Lineage^- Sca1^high cKit^high (LSK) and bipotential Pre-Megakaryocytic/Erythroid Progenitor (PreMegE) populations, coincides with a loss of erythroid (Ery) specification. Accordingly the PreMegE population can be prospectively separated into “pro-erythroid” and “pro-megakaryocyte” populations based on Runx1 P2 activity. Comparative gene expression analyses between Runx1 P2⁺ and P2^- populations indicated that levels of CD34 expression could substitute for P2 activity to distinguish these two cell populations in wild type (WT) bone marrow (BM). Prospective isolation of these two populations will enable the further investigation of molecular mechanisms involved in megakaryocytic/erythroid (Mk/Ery) cell fate decisions. Having characterized the extensive activity of P1, we utilized a P1-GFP homozygous mouse model to analyze the impact of the complete absence of Runx1 P1 expression in adult mice and observed strong defects in the T cell lineage. Finally, we investigated how the leukemic fusion protein AML1-ETO9a might influence Runx1 promoter usage. Short-term AML1-ETO9a induction in BM resulted in preferential P2 upregulation, suggesting its expression may be important to establish a pre-leukemic environment.     

Polymorphism in exon 6 of the human NT5E gene is associated with aortic valve calcification

ABSTRACT

NT5E encodes ecto-5′-nucleotidase (e5NT, CD73) which hydrolyses extracellular AMP to adenosine. Adenosine has been shown to play a protective role against aortic valve calcification (AVC). We identified two nonsynonymous missense single nucleotide polymorphisms (c.1126A > G, p.T376A and c.1136T > C, p.M379T) in exon 6 of the human NT5E gene. Since both substitutions might affect e5NT activity and consequently alter extracellular adenosine levels, we evaluated the association between NT5E alleles and calcific aortic valve disease in 119 patients (95 patients with AVC and 24 controls). In AVC patients, the frequency of the G allele at c.1126 and the frequency of the GG genotype as well as the frequency of the C allele at c.1136, and the frequencies of CC and TC genotypes tended to be higher as compared to controls. The allele and genotype frequencies in AVC patients and controls were also compared to those calculated from the 1000 Genomes Project data for control individuals of European ancestry (n = 503). We found that the frequency of the C allele at c.1136 is significantly higher in patients with AVC than in the European controls (0.111 vs. 0.054, P = 0.0052). Moreover, e5NT activity in aortic valves showed a trend toward lower levels in AVC patients with CC and TC genotypes than in those with the TT genotype. Our findings indicate that the genetic polymorphism of NT5E may contribute to the pathogenesis of calcific aortic valve disease and that the C allele of SNP c.1136 is associated with an increased risk of AVC.     

Redox conditions during sedimentation of the Middle Jurassic (Upper Bajocian–Bathonian) clays of the Polish Jura (south-central Poland)

Depositional redox conditions of the uppermost Bajocian–Bathonian (Middle Jurassic) ore-bearing clays of the Gnaszyn/Kawodrza area in the Polish Jura have been determined using an integrated geochemical (Th/U and U/Th ratios, degree of pyritisation (DOP), sulphur stable isotopes, biomarker analysis) and petrographic approach (measurements of pyrite framboid diameters, and microfacies analysis). The Th/U and U/Th ratios indicate that oxic conditions prevailed on the sea-floor during this interval, and ³⁴S isotopes suggest open-system conditions. DOP values, however, are rather scattered, and may reflect oxic, dysoxic, or anoxic conditions. We consider that the DOP values result from reducing conditions within the sediment and the chemistry of the pore-waters, rather than true sea-floor redox conditions. Pyrite framboid populations also indicate that dysoxic conditions prevailed within the sediment, beneath an oxygenated water column. Biomarker data did not provide any evidence of water column stratification or anoxia during sedimentation of the Middle Jurassic clays.