Influenza H5N1 virus infection of polarized human alveolar epithelial cells and lung microvascular endothelial cells

Background

Highly pathogenic avian influenza (HPAI) H5N1 virus is entrenched in poultry in Asia and Africa and continues to infect humans zoonotically causing acute respiratory disease syndrome and death. There is evidence that the virus may sometimes spread beyond respiratory tract to cause disseminated infection. The primary target cell for HPAI H5N1 virus in human lung is the alveolar epithelial cell. Alveolar epithelium and its adjacent lung microvascular endothelium form host barriers to the initiation of infection and dissemination of influenza H5N1 infection in humans. These are polarized cells and the polarity of influenza virus entry and egress as well as the secretion of cytokines and chemokines from the virus infected cells are likely to be central to the pathogenesis of human H5N1 disease.

Aim

To study influenza A (H5N1) virus replication and host innate immune responses in polarized primary human alveolar epithelial cells and lung microvascular endothelial cells and its relevance to the pathogenesis of human H5N1 disease.

Methods

We use an in vitro model of polarized primary human alveolar epithelial cells and lung microvascular endothelial cells grown in transwell culture inserts to compare infection with influenza A subtype H1N1 and H5N1 viruses via the apical or basolateral surfaces.

Results

We demonstrate that both influenza H1N1 and H5N1 viruses efficiently infect alveolar epithelial cells from both apical and basolateral surface of the epithelium but release of newly formed virus is mainly from the apical side of the epithelium. In contrast, influenza H5N1 virus, but not H1N1 virus, efficiently infected polarized microvascular endothelial cells from both apical and basolateral aspects. This provides a mechanistic explanation for how H5N1 virus may infect the lung from systemic circulation. Epidemiological evidence has implicated ingestion of virus-contaminated foods as the source of infection in some instances and our data suggests that viremia, secondary to, for example, gastro-intestinal infection, can potentially lead to infection of the lung. HPAI H5N1 virus was a more potent inducer of cytokines (e.g. IP-10, RANTES, IL-6) in comparison to H1N1 virus in alveolar epithelial cells, and these virus-induced chemokines were secreted onto both the apical and basolateral aspects of the polarized alveolar epithelium.

Conclusion

The predilection of viruses for different routes of entry and egress from the infected cell is important in understanding the pathogenesis of influenza H5N1 infection and may help unravel the pathogenesis of human H5N1 disease.     

ETISEQ – an algorithm for automated elution time ion sequencing of concurrently fragmented peptides for mass spectrometry-based proteomics

Background  

Concurrent peptide fragmentation (i.e. shotgun CID, parallel CID or MS^E) has emerged as an alternative to data-dependent acquisition in generating peptide fragmentation data in LC-MS/MS proteomics experiments. Concurrent peptide fragmentation data acquisition has been shown to be advantageous over data-dependent acquisition by providing greater detection dynamic range and providing more accurate quantitative information. Nevertheless, concurrent peptide fragmentation data acquisition remains to be widely adopted due to the lack of published algorithms designed specifically to process or interpret such data acquired on any mass spectrometer.     

Genetic Diversity and Population Structure Analysis of European Hexaploid Bread Wheat (Triticum aestivum L.) Varieties

Progress in plant breeding is facilitated by accurate information about genetic structure and diversity. Here, Diversity Array Technology (DArT) was used to characterize a population of 94 bread wheat (Triticum aestivum L.) varieties of mainly European origin. In total, 1,849 of 7,000 tested markers were polymorphic and could be used for population structure analysis. Two major subgroups of wheat varieties, GrI and GrII, were identified using the program STRUCTURE, and confirmed by principal component analysis (PCA). These subgroups were largely separated according to origin; GrI comprised varieties from Southern and Eastern Europe, whereas GrII contained mostly modern varieties from Western and Northern Europe. A large proportion of the markers contributing most to the genetic separation of the subgroups were located on chromosome 2D near the Reduced height 8 (Rht8) locus, and PCR-based genotyping suggested that breeding for the Rht8 allele had a major impact on subgroup separation. Consistently, analysis of linkage disequilibrium (LD) suggested that different selective pressures had acted on chromosome 2D in the two subgroups. Our data provides an overview of the allele composition of bread wheat varieties anchored to DArT markers, which will facilitate targeted combination of alleles following DArT-based QTL studies. In addition, the genetic diversity and distance data combined with specific Rht8 genotypes can now be used by breeders to guide selection of crossing parents.     

Blood Born miRNAs Signatures that Can Serve as Disease Specific Biomarkers Are Not Significantly Affected by Overall Fitness and Exercise

Blood born micro(mi)RNA expression pattern have been reported for various human diseases with signatures specific for diseases. To evaluate these biomarkers, it is mandatory to know possible changes of miRNA signatures in healthy individuals under different physiological conditions. We analyzed the miRNA expression in peripheral blood of elite endurance athletes and moderatly active controls. Blood drawing was done before and after exhaustive exercise in each group. After Benjamini-Hochberg adjustment we did not find any miRNA with significant p-values when comparing miRNA expression between the different groups. We found, however, 24 different miRNAs with an expression fold change of minimum 1.5 in at least one of the comparisons (athletes before vs after exercise, athletes before exercise vs controls and athletes after exercise vs controls). The observed changes are not significant in contrast to the expression changes of the blood born miRNA expression reported for many human diseases. These data support the idea of disease associated miRNA patterns useful as biomarkers that are not readily altered by physiological conditions.     

RFLP markers to identify the alleles on the Mla locus conferring powdery mildew resistance in barley

Summary To identify the mildew resistance locus Mla in barley with molecular markers, closely linked genomic RFLP clones were selected with the help of near-isogenic lines having the Pallas and Siri background. Out of 22 polymorphic clones 3 were located around the Mla locus on chromosome 5 with a distance of 5.1 + 2.9 cM (MWG 1H068), 4.2±1.7 cM (MWG 1H060) and 0.7 ± 0.7 cM (MWG 1H036), respectively. The polymorphic clone MWG 1H036 displayed the same RFLP pattern in both Pallas and Siri near-isogenic lines and in different varieties digested with six restriction enzymes possessing the same mildew resistance gene. The alleles of the Mla locus were grouped in 11 classes according to their specific RFLP patterns; 3 of these groups contain the majority of Mla alleles already used in barley breeding programs in Europe.     

Liquid Exfoliated Co(OH)2 Nanosheets as Low‐Cost,Yet High‐Performance,Catalysts for the Oxygen Evolution Reaction

Identifying cheap, yet effective, oxygen evolution catalysts is critical to the advancement of water splitting. Using liquid exfoliated Co(OH)₂ nanosheets as a model system, a simple procedure is developed to maximize the activity of any oxygen evolution reaction nanocatalyst. First the nanosheet edges are confirmed as the active areas by analyzing the catalytic activity as a function of nanosheet size. This allows the authors to select the smallest nanosheets (length ≈50 nm) as the best performing catalysts. While the number of active sites per unit electrode area can be increased via the electrode thickness, this is found to be impossible beyond ≈10 µm due to mechanical instabilities. However, adding carbon nanotubes increases both toughness and conductivity significantly. These enhancements mean that composite electrodes consisting of small Co(OH)₂ nanosheets and 10 wt% nanotubes can be made into freestanding films with thickness of up to 120 µm with no apparent electrical limitations. The presence of diffusion limitations results in an optimum electrode thickness of 70 µm, yielding a current density of 50 mA cm^?2 at an overpotential of 235 mV, close to the state of the art in the field. Applying this procedure to a high‐performance catalyst such as NiFeO_x should significantly surpass the state of the art.     

Genome-wide MicroRNA Expression Profiles in COPD: Early Predictors for Cancer Development

Chronic obstructive pulmonary disease(COPD) significantly increases the risk of developing cancer. Biomarker studies frequently follow a case-control set-up in which patients diagnosed with a disease are compared to controls. Longitudinal cohort studies such as the COPD-centered German COPD and SYstemic consequences-COmorbidities NETwork(COSYCONET) study provide the patient and biomaterial base for discovering predictive molecular markers. We asked whether microRNA(miRNA) profiles in blood collected from COPD patients prior to a tumor diagnosis could support an early diagnosis of tumor development independent of the tumor type. From 2741 participants of COSYCONET diagnosed with COPD, we selected 534 individuals including 33 patients who developed cancer during the follow-up period of 54 months and 501 patients who did not develop cancer, but had similar age, gender and smoking history. Genome-wide miRNA profiles were generated and evaluated using machine learning techniques. For patients developing cancer we identified nine miRNAs with significantly decreased abundance(two-tailed unpaired t-test adjusted for multiple testing P 0.05), including members of the miR-320 family. The identified mi RNAs regulate different cancer-related pathways including the MAPK pathway(P = 2.3 x 10~(-5)). We also observed the impact of confounding factors on the generated miRNA profiles, underlining the value of our matched analysis. For selected miRNAs, qRT-PCR analysis was applied to validate the results. In conclusion, we identified several miRNAs in blood of COPD patients, which could serve as candidates for biomarkers to help identify COPD patients at risk of developing cancer.     

Localization of quantitative trait loci (QTL) for agronomic important characters by the use of a RFLP map in barley (Hordeum vulgare L.)

Two hundred and fifty doubled haploid lines were studied from a cross between two 2-row winter barley varieties. The lines were evaluated for several characters in a field experiment for 3 years on two locations with two replications. From a total of 431 RFLP probes 50 were found to be polymorphic and subsequently used to construct a linkage map. Quantitative trait loci (QTLs) were determined and localized for resistance against Rhynchosporium secalis and Erysiphe graminis, for lodging, stalk breaking and ear breaking tendency, for the physical state before harvest, plant height, heading date, several kernel parameters and kernel yield. The heritability of the traits ranged from 0.56 to 0.89. For each trait except for kernel thickness, QTLs have been localized that explain 5–52% of the genetic variance. Transgressive segregation occurred for all of the traits studied.     

Glyoxalase-1 overexpression partially prevents diabetes-induced impaired arteriogenesis in a rat hindlimb ligation model

We hypothesize that diabetes-induced impaired collateral formation after a hindlimb ligation in rats is in part caused by intracellular glycation and that overexpression of glyoxalase-I (GLO-I), i.e. the major detoxifying enzyme for advanced-glycation-endproduct (AGE) precursors, can prevent this. Wild-type and GLO-I transgenic rats with or without diabetes (induced by 55 mg/kg streptozotocin) were subjected to ligation of the right femoral artery. Laser Doppler perfusion imaging showed a significantly decreased blood perfusion recovery after 6 days in the diabetic animals compared with control animals, without any effect of Glo1 overexpression. In vivo time-of-flight magnetic resonance angiography at 7-Tesla showed a significant decrease in the number and volume of collaterals in the wild-type diabetic animals compared with the control animals. Glo1 overexpression partially prevented this decrease in the diabetic animals. Diabetes-induced impairment of arteriogenic adaptation can be partially rescued by overexpressing of GLO-I, indicating a role of AGEs in diabetes-induced impaired collateral formation.     

Resonance Raman spectroscopy of ribonucleotide reductase. Evidence for a deprotonated tyrosyl radical and photochemistry of the binuclear iron center

Native ribonucleotide reductase from Escherichia coli exhibits a resonance-enhanced Raman mode at 1498 cm-1 that is characteristic of a tyrosyl radical. The Raman frequency as well as the absorption maximum at 410 nm identifies the radical as being in a deprotonated state. The B2 subunit of ribonucleotide reductase shows an additional resonance Raman mode at 493 cm-1 that has been assigned to the symmetric stretch of an Fe-O-Fe moiety. When samples of active B2 or metB2 are exposed to a tightly focused laser beam at 406.7 nm, there is a loss of intensity at 493 cm-1 and the appearance of a new peak at 595 cm-1. Although the 595-cm-1 feature was previously assigned to an Fe-OH vibration on the basis of its 23-cm-1 shift to lower energy in H2(18)O and the apparent dependence of its intensity on pH [Sj?berg, B. M., Loehr, T. M., & Sanders-Loehr, J. (1987) Biochemistry 26, 4242], the present studies indicate that the intensity of this mode is dependent primarily on input laser power. The peak at 595 cm-1 is more plausibly assigned to a new vs(Fe-O-Fe) mode in view of its lack of the deuterium isotope dependence expected for an Fe-OH mode and its resonant scattering cross section which is comparable to that of the 493-cm-1 mode. This new species has a calculated Fe-O-Fe angle of approximately 113 degrees compared to approximately 138 degrees calculated for the Fe-O-Fe unit in unmodified protein B2. One possible explanation for the photoinduced vibrational mode is that a bridging solvent molecule has been inserted in place of a bridging carboxylate.