Thymidine Kinases in Archaea

Twenty-six fully sequenced archaeal genomes were searched for genes coding for putative deoxyribonucleoside kinases (dNKs). We identified only 5 human-like thymidine kinase 1 genes (TK1s) and none for non-TK1 kinases. Four TK1s were identified in the Euryarchaea and one was found in the Crenarchaea, while none was found in Nanoarchaeum. The identified TK1s have high identity to Gram-positive bacteria TK1s. The TK1s from archaea, Gram-positive bacteria and eukaryotes share the same common ancestor, while the TK1s from Gram-negative bacteria belong to a less-related subgroup. It seems that a functional deoxyribonucleoside salvage pathway is not crucial for the archaeal cell.     

Niche Divergence versus Neutral Processes: Combined Environmental and Genetic Analyses Identify Contrasting Patterns of Differentiation in Recently Diverged Pine Species

Background and Aims

Solving relationships of recently diverged taxa, poses a challenge due to shared polymorphism and weak reproductive barriers. Multiple lines of evidence are needed to identify independently evolving lineages. This is especially true of long-lived species with large effective population sizes, and slow rates of lineage sorting. North American pines are an interesting group to test this multiple approach. Our aim is to combine cytoplasmic genetic markers with environmental information to clarify species boundaries and relationships of the species complex of Pinus flexilis, Pinus ayacahuite, and Pinus strobiformis.

Methods

Mitochondrial and chloroplast sequences were combined with previously obtained microsatellite data and contrasted with environmental information to reconstruct phylogenetic relationships of the species complex. Ecological niche models were compared to test if ecological divergence is significant among species.

Key Results and Conclusion

Separately, both genetic and ecological evidence support a clear differentiation of all three species but with different topology, but also reveal an ancestral contact zone between P. strobiformis and P. ayacahuite. The marked ecological differentiation of P. flexilis suggests that ecological speciation has occurred in this lineage, but this is not reflected in neutral markers. The inclusion of environmental traits in phylogenetic reconstruction improved the resolution of internal branches. We suggest that combining environmental and genetic information would be useful for species delimitation and phylogenetic studies in other recently diverged species complexes.     

Microbial Environment Affects Innate Immunity in Two Closely Related Earthworm Species Eisenia andrei and Eisenia fetida

Survival of earthworms in the environment depends on their ability to recognize and eliminate potential pathogens. This work is aimed to compare the innate defense mechanisms of two closely related earthworm species, Eisenia andrei and Eisenia fetida, that inhabit substantially different ecological niches. While E. andrei lives in a compost and manure, E. fetida can be found in the litter layer in forests. Therefore, the influence of environment-specific microbiota on the immune response of both species was followed. Firstly, a reliable method to discern between E. andrei and E. fetida based on species-specific primers for cytochrome c oxidase I (COI) and stringent PCR conditions was developed. Secondly, to analyze the immunological profile in both earthworm species, the activity and expression of lysozyme, pattern recognition protein CCF, and antimicrobial proteins with hemolytic function, fetidin and lysenins, have been assessed. Whereas, CCF and lysozyme showed only slight differences in the expression and activity, fetidin/lysenins expression as well as the hemolytic activity was considerably higher in E. andrei as compared to E. fetida. The expression of fetidin/lysenins in E. fetida was not affected upon the challenge with compost microbiota, suggesting more substantial changes in the regulation of the gene expression. Genomic DNA analyses revealed significantly higher level of fetidin/lysenins (determined using universal primer pairs) in E. andrei compared to E. fetida. It can be hypothesized that E. andrei colonizing compost as a new habitat acquired an evolutionary selection advantage resulting in a higher expression of antimicrobial proteins.     

Induction of Mouse Melioidosis with Meningitis by CD11b+ Phagocytic Cells Harboring Intracellular B. pseudomallei as a Trojan Horse

Background

Approximately 3–5% of patients with melioidosis manifest CNS symptoms; however, the clinical data regarding neurological melioidosis are limited.

Methods and Findings

We established a mouse model of melioidosis with meningitis characterized by neutrophil infiltration into the meninges histologically and B. pseudomallei in the cerebrospinal fluid (CSF) by bacteriological culturing methods. As the disease progresses, the bacteria successively colonize the spleen, liver, bone marrow (BM) and brain and invade splenic and BM cells by days 2 and 6 post-infection, respectively. The predominant cell types intracellularly infected with B. pseudomallei were splenic and BM CD11b⁺ populations. The CD11b⁺Ly6C^high inflamed monocytes, CD11b⁺Ly6C^low resident monocytes, CD11b⁺Ly6G⁺ neutrophils, CD11b⁺F4/80⁺ macrophages and CD11b⁺CD19⁺ B cells were expanded in the spleen and BM during the progression of melioidosis. After adoptive transfer of CD11b populations harboring B. pseudomallei, the infected CD11b⁺ cells induced bacterial colonization in the brain, whereas CD11b⁻ cells only partially induced colonization; extracellular (free) B. pseudomallei were unable to colonize the brain. CD62L (selectin) was absent on splenic CD11b⁺ cells on day 4 but was expressed on day 10 post-infection. Adoptive transfer of CD11b⁺ cells expressing CD62L (harvested on day 10 post-infection) resulted in meningitis in the recipients, but transfer of CD11b⁺ CD62L-negative cells did not.

Conclusions/Significance

We suggest that B. pseudomallei-infected CD11b⁺ selectin-expressing cells act as a Trojan horse and are able to transmigrate across endothelial cells, resulting in melioidosis with meningitis.     

Valproic Acid Treatment Inhibits Hypoxia-Inducible Factor 1α Accumulation and Protects against Burn-Induced Gut Barrier Dysfunction in a Rodent Model

Objective

Burn-induced gut dysfunction plays an important role in the development of sepsis and multiple organ dysfunction. Emerging evidence suggests that hypoxia-inducible factor-1α (HIF-1α) is critical in paracelluar barrier functions via regulating vascular endothelial growth factor (VEGF) and myosin light chain kinase (MLCK) expression. Previous studies have also demonstrated that histone deacetylase inhibitors (HDACIs) can repress HIF-1α. This study aims to examine whether valproic acid (VPA), a HDACI, protects against burn-induced gut barrier dysfunction via repressing HIF-1α-dependent upregulation of VEGF and MLCK expression.

Methods

Rats were subjected to third degree 55% TBSA burns and treated with/ without VPA (300mg/kg). Intestinal barrier dysfunction was evaluated by permeability of intestinal mucosa to fluorescein isothiocyanate (FITC)-dextran and histologic evaluation. Histone acetylation, tight junction protein zonula occludens 1 (ZO-1), VEGF, MLCK and HIF-1α were measured. In addition, CaCO₂ cells were transfected with siRNA directed against HIF-1α and were stimulated with CoCl2 (1mM) for 24 hours with/without VPA (2mM) followed by analysis of HIF-1α, MLCK, VEGF and ZO-1.

Results

Burn insults resulted in a significant increase in intestinal permeability and mucosal damage, accompanied by a significant reduction in histone acetylation, ZO-1, upregulation of VEGF, MLCK expression, and an increase in HIF-1α accumulation. VPA significantly attenuated the increase in intestinal permeability, mucosa damage, histone deacetylation and changes in ZO-1 expression. VPA also attenuated the increased VEGF, MLCK and HIF-1α protein levels. VPA reduced HIF-1α, MLCK and VEGF production and prevented ZO-1 loss in CoCl2-stimulated Caco-2 cells. Moreover, transfection of siRNA directed against HIF-1α led to inhibition of MLCK and VEGF production, accompanied by upregulation of ZO-1.

Conclusions

These results indicate that VPA can protect against burn-induced gut barrier dysfunction. These protective effects may be due to its inhibitory action on HIF-1α, leading to a reduction in intestinal VEGF and MLCK expression and minimizing ZO-1 degradation.     

The shift from low to high non-structural protein 1 expression in rotavirus-infected MA-104 cells

A hallmark of group/species A rotavirus (RVA) replication in MA-104 cells is the logarithmic increase in viral mRNAs that occurs four-12 h post-infection. Viral protein synthesis typically lags closely behind mRNA synthesis but continues after mRNA levels plateau. However, RVA non-structural protein 1 (NSP1) is present at very low levels throughout viral replication despite showing robust protein synthesis. NSP1 has the contrasting properties of being susceptible to proteasomal degradation, but being stabilised against proteasomal degradation by viral proteins and/or viral mRNAs. We aimed to determine the kinetics of the accumulation and intracellular distribution of NSP1 in MA-104 cells infected with rhesus rotavirus (RRV). NSP1 preferentially localises to the perinuclear region of the cytoplasm of infected cells, forming abundant granules that are heterogeneous in size. Late in infection, large NSP1 granules predominate, coincident with a shift from low to high NSP1 expression levels. Our results indicate that rotavirus NSP1 is a late viral protein in MA-104 cells infected with RRV, presumably as a result of altered protein turnover.     

Enhanced production of N-acetyl-d-neuraminic acid by multi-approach whole-cell biocatalyst

N-Acetyl-d-neuraminic acid (Neu5Ac) has attracted considerable interest due to its promising potential applications in medicine. Significant efforts have been made in whole-cell biocatalyst for Neu5Ac production, but the processes often result in suboptimal performance due to poor expression of enzymes, imbalances of pathway components, disturbance of competing pathways, and barriers of mass transport. In this study, we engineered Escherichia coli strains capable of producing Neu5Ac by assembling a two-step heterologous pathway consisting of N-acetyl-d-glucosamine 2-epimerase (AGE) and Neu5Ac aldolase (NanA). Multiple approaches were used to improve the efficiency of the engineered pathway and process for enhanced Neu5Ac production. Firstly, we identified that NanA was the rate-controlling enzyme in this pathway. With increased expression of NanA, a ninefold increase in Neu5Ac production (65 mM) was observed. Secondly, knocking out nanTEK genes blocked Neu5Ac uptake and the competing pathway, which kept the reactions to the synthetic direction as the final product went outside of the cells and enhanced the Neu5Ac production by threefold, resulting in 173.8 mM of Neu5Ac. Thirdly, we improved the performance of the system by promoting substrate transport and optimizing concentrations of substrates. An overall whole-cell biocatalytic process was developed and a maximum titer of 240 mM Neu5Ac (74.2 g/L) was achieved, with productivity of 6.2 g Neu5Ac/L/h and conversion yield of 40 % from GlcNAc. The engineered strain could be reused for at least five cycles with a productivity of >6 g/L/h. It is a cost-effective process for Neu5Ac production with potential applications in large-scale industrial production.     

Revised and updated checklist of lichens of Slovakia (May 2013)

We characterize lichen flora of Slovakia based on the review of recent studies published since 1998, point at selected lichen taxa described for science from this territory and outline main geographical elements and other important groups. We annex critically revised and updated checklist of lichens of Slovakia, which includes 1,628 species. As a result of intensive field work the number of species increased by 142 new species since the last version of checklist published 15 years ago, 26 species were excluded.     

Functional,structural and phylogenetic analysis of domains underlying the Al sensitivity of the aluminum‐activated malate/anion transporter,TaALMT1

Triticum aestivum aluminum‐activated malate transporter (TaALMT1) is the founding member of a unique gene family of anion transporters (ALMTs) that mediate the efflux of organic acids. A small sub‐group of root‐localized ALMTs, including TaALMT1, is physiologically associated with in planta aluminum (Al) resistance. TaALMT1 exhibits significant enhancement of transport activity in response to extracellular Al. In this study, we integrated structure–function analyses of structurally altered TaALMT1 proteins expressed in Xenopus oocytes with phylogenic analyses of the ALMT family. Our aim is to re‐examine the role of protein domains in terms of their potential involvement in the Al‐dependent enhancement (i.e. Al‐responsiveness) of TaALMT1 transport activity, as well as the roles of all its 43 negatively charged amino acid residues. Our results indicate that the N‐domain, which is predicted to form the conductive pathway, mediates ion transport even in the absence of the C‐domain. However, segments in both domains are involved in Al³⁺ sensing. We identified two regions, one at the N‐terminus and a hydrophobic region at the C‐terminus, that jointly contribute to the Al‐response phenotype. Interestingly, the characteristic motif at the N‐terminus appears to be specific for Al‐responsive ALMTs. Our study highlights the need to include a comprehensive phylogenetic analysis when drawing inferences from structure–function analyses, as a significant proportion of the functional changes observed for TaALMT1 are most likely the result of alterations in the overall structural integrity of ALMT family proteins rather than modifications of specific sites involved in Al³⁺ sensing.