The spore‐associated protein BclA1 affects the susceptibility of animals to colonization and infection by Clostridium difficile

The BclA protein is a major component of the outermost layer of spores of a number of bacterial species and Clostridium difficile carries three bclA genes. Using insertional mutagenesis each gene was characterized and spores devoid of these proteins had surface aberrations, reduced hydrophobicity and germinated faster than wild‐type spores. Therefore the BclA proteins were likely major components of the spore surface and when absent impaired the protective shield effect of this outermost layer. Analysis of infection and colonization in mice and hamsters revealed that the 50% infectious dose (ID₅₀) of spores was significantly higher (2‐logs) in the bclA1^? mutant compared to the isogenic wild‐type control, but that levels of toxins (A and B) were indistinguishable from animals dosed with wild‐type spores. bclA1^? spores germinated faster than wild‐type spores yet mice were less susceptible to infection suggesting that BclA1 must play a key role in the initial (i.e. pre‐spore germination) stages of infection. We also show that the ID₅₀ was higher in mice infected with R20291, a ‘hypervirulent’ 027 strain, that carries a truncated BclA1 protein.     

Synthetic substrates for measuring activity of autophagy proteases: Autophagins (Atg4)

Atg4 cysteine proteases (autophagins) play crucial roles in autophagy by proteolytic activation of Atg8 paralogs for targeting to autophagic vesicles by lipid conjugation, as well as in subsequent deconjugation reactions. However, the means to measure the activity of autophagins is limited. Herein, we describe two novel substrates for autophagins suitable for a diversity of in vitro assays, including (i) fluorogenic tetrapeptide acetyl-Gly-L-Thr-L-Phe-Gly-AFC (Ac-GTFG-AFC) and (ii) a fusion protein comprised of the natural substrate LC3B appended to the N-terminus of phospholipase A₂ (LC3B-PLA₂), which upon cleavage releases active PLA₂ for fluorogenic assay. To generate the synthetic tetrapeptide substrate, the preferred tetrapeptide sequence recognized by autophagin-1/Atg4B was determined using a positional scanning combinatorial fluorogenic tetrapeptide library. With the LC3B-PLA₂ substrate, we show that mutation of the glycine proximal to the scissile bond in LC3B abolishes activity. Both substrates showed high specificity for recombinant purified autophagin-1/Atg4B compared to closely related proteases and the LC3B-PLA₂ substrate afforded substantially higher catalytic rates (k_cat/K_m 5.26 × 10⁵ M⁻¹/sec⁻¹) than Ac-GTFG-AFC peptide (0.92 M⁻¹/sec⁻¹), consistent with substrate-induced activation. Studies of autophagin-1 mutants were also performed, including the protease lacking a predicted autoinhibitory domain at residues 1 to 24 and lacking a regulatory loop at residues 259 to 262. The peptide and fusion protein substrates were also employed for measuring autophagin activity in cell lysates, showing a decrease in cells treated with autophagin-1/Atg4B siRNA or transfected with a plasmid encoding Atg4B (Cys74Ala) dominant-negative. Therefore, the synthetic substrates for autophagins reported here provide new research tools for studying autophagy.Key words: autophagin, fluorogenic assay, tetrapeptide, phospholipase A2, LC3     

Lecithin:retinol acyltransferase is critical for cellular uptake of vitamin A from serum retinol-binding protein

Vitamin A (all-trans-retinol) must be adequately distributed within the mammalian body to produce visual chromophore in the eyes and all-trans-retinoic acid in other tissues. Vitamin A is transported in the blood bound to retinol-binding protein (holo-RBP), and its target cells express an RBP receptor encoded by the Stra6 (stimulated by retinoic acid 6) gene. Here we show in mice that cellular uptake of vitamin A from holo-RBP depends on functional coupling of STRA6 with intracellular lecithin:retinol acyltransferase (LRAT). Thus, vitamin A uptake from recombinant holo-RBP exhibited by wild type mice was impaired in Lrat(-/-) mice. We further provide evidence that vitamin A uptake is regulated by all-trans-retinoic acid in non-ocular tissues of mice. When in excess, vitamin A was rapidly taken up and converted to its inert ester form in peripheral tissues, such as lung, whereas in vitamin A deficiency, ocular retinoid uptake was favored. Finally, we show that the drug fenretinide, used clinically to presumably lower blood RBP levels and thus decrease circulating retinol, targets the functional coupling of STRA6 and LRAT to increase cellular vitamin A uptake in peripheral tissues. These studies provide mechanistic insights into how vitamin A is distributed to peripheral tissues in a regulated manner and identify LRAT as a critical component of this process.     

Carbon uptake in Eurasian boreal forests dominates the high-latitude net ecosystem carbon budget

Arctic-boreal landscapes are experiencing profound warming, along with changes in ecosystem moisture status and disturbance from fire. This region is of global importance in terms of carbon feedbacks to climate, yet the sign (sink or source) and magnitude of the Arctic-boreal carbon budget within recent years remains highly uncertain. Here, we provide new estimates of recent (2003–2015) vegetation gross primary productivity (GPP), ecosystem respiration (R_eco), net ecosystem CO₂ exchange (NEE; R_eco − GPP), and terrestrial methane (CH₄) emissions for the Arctic-boreal zone using a satellite data-driven process-model for northern ecosystems (TCFM-Arctic), calibrated and evaluated using measurements from >60 tower eddy covariance (EC) sites. We used TCFM-Arctic to obtain daily 1-km² flux estimates and annual carbon budgets for the pan-Arctic-boreal region. Across the domain, the model indicated an overall average NEE sink of −850 Tg CO₂-C year⁻¹. Eurasian boreal zones, especially those in Siberia, contributed to a majority of the net sink. In contrast, the tundra biome was relatively carbon neutral (ranging from small sink to source). Regional CH₄ emissions from tundra and boreal wetlands (not accounting for aquatic CH₄) were estimated at 35 Tg CH₄-C year⁻¹. Accounting for additional emissions from open water aquatic bodies and from fire, using available estimates from the literature, reduced the total regional NEE sink by 21% and shifted many far northern tundra landscapes, and some boreal forests, to a net carbon source. This assessment, based on in situ observations and models, improves our understanding of the high-latitude carbon status and also indicates a continued need for integrated site-to-regional assessments to monitor the vulnerability of these ecosystems to climate change.     

Seed morphology and endosperm structure of selected species of Primulaceae, Myrsinaceae, and Theophrastaceae and their systematic importance

Seed size and shape, seed coat surface pattern, seed coat thickness, and endosperm structure were investigated in Androsace septentrionalis, Cortusa matthioli, Hottonia palustris, Primula elatior, Soldanella carpatica (Primulaceae), Anagallis arvensis, A. minima, Cyclamen purpurascens, Glaux maritima, Lysimachia nemorum, L. vulgaris, Trientalis europaea (Myrsinaceae), and Samolus valerandi (Theophrastaceae). Three seed size categories were distinguished on the basis of biometric measurements. Almost all seeds examined were found to be small with an angular shape classified as sectoroid or polyhedral. A new type of seed shape, suboval, was identified for H. palustris. Cyclamen purpurascens seeds differed from seeds of all other species examined because of their large size, subglobose shape, and concave hilar area. Despite the different shape types, the length/width ratio of the seeds examined was constant, while their hilum length/width ratio was a highly variable feature. Three types of seed surface patterns were observed: (1) reticulate, (2) tuberculate with secondary striations, and (3) poroid-alveolate with the presence of a spongy outer layer. For seeds of Anagallis arvensis, A. minima, Cortusa matthioli, Lysimachia nemorum, and Soldanella carpatica, secondary seed sculpture was described. The seed coats of all species examined were two-layered, and great differences in testa thickness were found (9.9?C128.6???m). In general, seeds of the Myrsinaceae species were more often characterized by thick testa. Different proportions in thickness of the inner and outer testa layers were observed. In seeds with reticulate seed patterns, the inner testa layer was twice to several times thicker than the outer layer, while the opposite proportions were observed in seeds with the tuberculate or poroid-alveolate seed sculpture pattern. In seeds of all species examined, oxalate crystals were present on the surface of the inner testa layer. Thus, the presence or absence of oxalate crystals in testa is not a feature distinguishing species with angular or subglobose seeds. Four types of endosperm structure were identified according to the thickness of the endosperm cell walls and the relief of their inner surface: (1) with evenly thickened and smooth cell walls, (2) with evenly thickened cell walls and circular or helical thickenings on their inside surfaces, (3) with very thick, but not evenly thickened cell walls with constrictions (??pitted??), and (4) with very thin papery and undulate cell walls. There is no rule concerning the seed shape, type of the seed sculpture, testa thickness, or endosperm structure that corresponds to the family affiliation of the species examined.     

A critical comparative assessment of predictions of protein-binding sites for biologically relevant organic compounds

Protein function annotation and rational drug discovery rely on the knowledge of binding sites for small organic compounds, and yet the quality of existing binding site predictors was never systematically evaluated. We assess predictions of ten representative geometry-, energy-, threading-, and consensus-based methods on a new benchmark data set that considers apo and holo protein structures with multiple binding sites for biologically relevant ligands. Statistical tests show that threading-based Findsite outperforms other predictors when its templates have high similarity with the input protein. However, Findsite is equivalent or inferior to some geometry-, energy-, and consensus-based methods when the similarity is lower. We demonstrate that geometry-, energy-, and consensus-based predictors benefit from the usage of holo structures and that the top four methods, Findsite, Q-SiteFinder, ConCavity, and MetaPocket, perform better for larger binding sites. Predictions from these four methods are complementary, and our simple meta-predictor improves over the best single predictor.     

Differential expression of Snail1 transcription factor and Snail1-related genes in alveolar and embryonal rhabdomyosarcoma subtypes

Rhabdomyosarcoma (RMS) represents the most common sarcoma of soft tissue among children. Two main RMS subtypes are alveolar (ARMS) and embryonal (ERMS). The major goal of this study was to find differentially expressed genes between RMS subtypes that could explain higher metastatic potential in ARMS and would be useful for the differential diagnosis. Using RQ-PCR analysis we compared expression of Snail1 and Snail-related genes among 7 ARMS and 8 ERMS patients' samples obtained from the primary tumors and among 2 alveolar and 2 embryonal cell lines. Our results show that Snail1 is highly expressed both in ARMS patients' samples and the alveolar cell lines. We also found that the expression of E-Cadherin was downregulated and the expression of Matrix Metalloproteinases 2 and 9 (MMP-2 and MMP-9) was upregulated in ARMS. We assume that, as in many tumors, also in RMS Snail1 acts as a regulator for pathways known for their role in cells' metastasis and that Snail1 activity results in increased MMPs and decreased E-Cadherin expression. Our findings may explain higher ARMS aggressiveness. Moreover, we suggest that further studies should be performed to verify if Snail1 can be considered as a potential target for ARMS therapy.     

Coordination of iron ions in the form of histidinyl dinitrosyl complexes does not prevent their genotoxicity

Formation of dinitrosyl iron complexes (DNICs) was observed in a wide spectrum of pathophysiological conditions associated with overproduction of NO. To gain insight into the possible genotoxic effects of DNIC, we examined the interaction of histidinyl dinitrosyl iron complexes (HIS-DNIC) with DNA by means of circular dichroism. Formation of DNIC was monitored by EPR and FT/IR spectroscopy. Vibrational bands for aquated HIS-DNIC are reported. Dichroism results indicate that HIS-DNIC changes the conformation of the DNA in a dose-dependent manner in 10 mM phosphate buffer (pH 6). Increase of the buffer pH or ionic strength decreased the effect. Comparison of HIS-DNIC DNA interaction with the effect of hydrated Fe²⁺ ion revealed many similarities. The importance of iron ions in HIS-DNIC induced genotoxicity is confirmed by plasmid nicking assay. Treatment of pUC19 plasmid with 1 μM HIS-DNIC did not affect the plasmid supercoiling. Higher concentrations of HIS-DNIC induced single strand breaks. The effect was completely abrogated by addition of deferoxamine, a specific strong iron chelator. Our data reveal that formation of HIS-DNIC does not prevent DNA from iron-induced damage and imply that there is no direct interrelationship between iron–NO coordination and their mutual toxicity modulation.     

Development of a PCR-based assay for rapid detection of class IIa bacteriocin genes

Aims: We have developed a PCR‐based assay using custom designed panel of primers which allows rapid detection of class IIa bacteriocin‐coding genes. To demonstrate the applicability of the developed assay, the method was applied on 40 metagenomic DNA preparations isolated from native microbiota of Polish artisanal cheeses produced in the Tatra Mountains. Methods and Results: The developed assay was designed on the basis of a large scale alignment of class IIa bacteriocin‐coding genes. A panel of seven primer pairs with confirmed ability to detect class IIa bacteriocin‐coding sequences was obtained. The following study has revealed a superb bacteriocinogenic potential of all forty analysed cheese samples. Conclusions: The majority of obtained sequences were lactic acid bacteria (LAB) related, although some sequences showed significant similarity to bacteriocin‐coding sequences present in non‐LAB bacteriocin producers. The results suggest that several potentially new bacteriocin‐coding sequences were found. Significance and Impact of the Study: The developed assay can be extremely helpful in establishing whether isolates from the environment of interest have a potential of synthesizing antilisterial class IIa bacteriocins. Application of the approach may represent a useful tool contributing to ecological studies looking for valuable probiotic, bacteriocinogenic microbiota developing in foods.