Nrf2 protects human alveolar epithelial cells against injury induced by influenza A virus

Background

Influenza A virus (IAV) infection primarily targets respiratory epithelial cells and produces clinical outcomes ranging from mild upper respiratory infection to severe pneumonia. Recent studies have shown the importance of lung antioxidant defense systems against injury by IAV. Nuclear factor-erythroid 2 related factor 2 (Nrf2) activates the majority of antioxidant genes.

Methods

Alveolar type II (ATII) cells and alveolar macrophages (AM) were isolated from human lungs not suitable for transplantation and donated for medical research. In some studies ATII cells were transdifferentiated to alveolar type I-like (ATI-like) cells. Alveolar epithelial cells were infected with A/PR/8/34 (PR8) virus. We analyzed PR8 virus production, influenza A nucleoprotein levels, ROS generation and expression of antiviral genes. Immunocytofluorescence was used to determine Nrf2 translocation and western blotting to detect Nrf2, HO-1 and caspase 1 and 3 cleavage. We also analyzed ingestion of PR8 virus infected apoptotic ATII cells by AM, cytokine levels by ELISA, glutathione levels, necrosis and apoptosis by TUNEL assay. Moreover, we determined the critical importance of Nrf2 using adenovirus Nrf2 (AdNrf2) or Nrf2 siRNA to overexpress or knockdown Nrf2, respectively.

Results

We found that IAV induced oxidative stress, cytotoxicity and apoptosis in ATI-like and ATII cells. We also found that AM can ingest PR8 virus-induced apoptotic ATII cells (efferocytosis) but not viable cells, whereas ATII cells did not ingest these apoptotic cells. PR8 virus increased ROS production, Nrf2, HO-1, Mx1 and OAS1 expression and Nrf2 translocation to the nucleus. Nrf2 knockdown with siRNA sensitized ATI-like cells and ATII cells to injury induced by IAV and overexpression of Nrf2 with AdNrf2 protected these cells. Furthermore, Nrf2 overexpression followed by infection with PR8 virus decreased virus replication, influenza A nucleoprotein expression, antiviral response and oxidative stress. However, AdNrf2 did not increase IFN-λ1 (IL-29) levels.

Conclusions

Our results indicate that IAV induces alveolar epithelial injury and that Nrf2 protects these cells from the cytopathic effects of IAV likely by increasing the expression of antioxidant genes. Identifying the pathways involved in protecting cells from injury during influenza infection may be particularly important for developing new therapeutic strategies.     

Influence of temperature on the biological parameters of the anholocyclic species Cinara tujafilina (Hemiptera: Aphidoidea)

Aphids are a good model to study insect reaction to habitat change. Temperature is one of the main factors that influences insects. This paper examines the influence of temperature on developmental stages, fecundity, survival rate and demographic parameters of Cinara tujafilina (Hemiptera: Aphidoidea, Lachnidae), connected with decorative plants of the Cupressaceae family. C. tujafilina was reared in a laboratory on T. orientalis at five constant temperatures of 10, 15, 20, 25 and 28°C, 70% humidity and 14L:10D. The pre-reproduction stage varied from 7 at 25°C to 19 days at 10°C. Developmental threshold was assigned at 3.5°C. The longest reproduction stage for the aphids developing was recorded at 25°C, namely 33 days, while the shortest, at the temperature of 10°C, lasted 8 days. At 25°C this species is characterised by the shortest pre-reproduction stage, the highest fecundity, the highest survival rate and the highest demographic parameters, particularly r_m (0.17). The results suggest that the optimal temperature for the species is 25°C, and indicate that climatic change will favourably influence its development and increase its role as a pest of decorative plants.     

Temporin A and its retro-analogues: synthesis, conformational analysis and antimicrobial activities.

Temporin A (TA) is a hydrophobic peptide isolated from the skin of the European red frog Rana temporaria. Strong antimicrobial activity against gram-positive cocci and Candida, as well as its small molecular weight (10-13 aa residues), makes TA an interesting antimicrobial compound. However, its synthesis is rather problematic. Here, the synthesis of two retro-analogues of TA--retro-TA and (6-1)(7-13)-TA--is reported. The synthesis of retro-TA was performed without any problems, while during the synthesis of (6-1)(7-13)-TA problems similar to those encountered during the synthesis of TA were faced. Antimicrobial assays showed minimal inhibitory concentration (MIC) values of retro-TA to be, in most cases, only one dilution higher than those of original TA, but still remained relatively low. An analysis of the circular dichroism spectra of the peptides shows that TA and (6-1)(7-13)-TA adopt an alpha-helical structure in a hydrophobic environment, while retro-TA forms mainly unordered conformation under both hydrophobic and hydrophilic conditions. One can postulate that differences in conformation of the peptide chain might be responsible for the lower antimicrobial activity of retro-TA as compared to that of the parent molecule. In any case, retro-TA can be interesting owing to its simple and nonproblematic synthesis.     

Redox reaction between amino-(3,4-dihydroxyphenyl)methyl phosphonic acid and dopaquinone is responsible for the apparent inhibitory effect on tyrosinase.

Amino-(3,4-dihydroxyphenyl)methyl phosphonic acid, the phosphonic analog of 3,4-dihydroxyphenylglycine, had been previously reported as a potent inhibitor of tyrosinase. The mechanism of the apparent enzyme inhibition by this compound has now been established. Amino-(3,4-dihydroxyphenyl)methyl phosphonic acid turned out to be a substrate and was oxidized to o-quinone, which evolved to a final product identified as 3,4-dihydroxybenzaldehyde, the same as for 3,4-dihydroxyphenylglycine. Monohydroxylated compounds (amino-(3-hydroxyphenyl)methyl phosphonic acid and amino-(4-hydroxyphenyl)methyl phosphonic acid) were not oxidized, neither was 4-hydroxy-l-phenylglycine. However, the relatively high Km for amino-(3,4-dihydroxyphenyl)methyl phosphonic acid (0.52 mm) indicated that competitive inhibition could not entirely explain the previously reported strong inhibitory effect (Ki = 50 and 97 micro m for tyrosine and 3-(3,4-dihydroxyphenyl)alanine (Dopa) as substrates, respectively). Neither was the enzyme covalently inactivated to a significant degree. Spectroscopic and electrochemical analysis of the oxidation of a mixture of Dopa and the inhibitor demonstrated that the phosphonic compound reduced dopaquinone back to Dopa, thus diminishing and delaying the formation of dopachrome. This produces an apparent strong inhibitory effect when the reaction is monitored spectrophotometrically at 475 nm. In this peculiar case Dopa acts as a redox shuttle mediating the oxidation of the shorter phosphonic homolog. Decomposition of the phosphonic o-quinone to 3,4-dihydroxybenzaldehyde drives the reaction against the slightly unfavorable difference in redox potentials.     

Towards a theory without adjacency: hyper-contextual VI-rules

It has been proposed that allomorphy and suppletion is restricted not only by (various conceptions of) cyclic locality, but also by adjacency of elements. Embick (2010) proposes that two elements can only enter into an relationship of allomorphy if they are linearly adjacent to each other, whereas Adger et al. (2003) and Bobaljik (2012) argue that elements must be structurally adjacent.In this paper, we show that there are both empirical and conceptual problems for adjacency based theories and we argue instead in favour of a cyclic locality only view; whereby allomorphic relations are not universally sensitive to adjacency restrictions. Apparent arguments in favour of adjacency are shown to be the result accessibility or of ‘hyper-contextual’ rules of vocabulary insertion, which make reference to multiple nodes, which combine together to serve as the context for suppletion.     

Visible‐Light Microscopic Discovery of Up to 150 μm Long Helical Amyloid Fibrils Built of the Dodecapeptide H‐(Val‐Ala‐Leu)4‐OH and of Decapeptides Derived from Insulin

In the formation of amyloid fibrils from small peptides, the appearance of superhelices of (P)‐ or (M)‐helicity has been observed for the first time; high concentrations of the peptides and extended periods of incubation at physiological pH appear to be important for this phenomenon. In view of the general importance of peptide and protein aggregation, we give a brief overview with selected examples for demonstration.