Deviating the level of proliferating cell nuclear antigen in Trypanosoma brucei elicits distinct mechanisms for inhibiting proliferation and cell cycle progression

The DNA replication machinery is spatially and temporally coordinated in all cells to reproduce a single exact copy of the genome per division, but its regulation in the protozoan parasite Trypanosoma brucei is not well characterized. We characterized the effects of altering the levels of proliferating cell nuclear antigen, a key component of the DNA replication machinery, in bloodstream form T. brucei. This study demonstrated that tight regulation of TbPCNA levels was critical for normal proliferation and DNA replication in the parasite. Depleting TbPCNA mRNA reduced proliferation, severely diminished DNA replication, arrested the synthesis of new DNA and caused the parasites to accumulated in G2/M. Attenuating the parasite by downregulating TbPCNA caused it to become hypersensitive to hydroxyurea. Overexpressing TbPCNA in T. brucei arrested proliferation, inhibited DNA replication and prevented the parasite from exiting G2/M. These results indicate that distinct mechanisms of cell cycle arrest are associated with upregulating or downregulating TbPCNA. The findings of this study validate deregulating intra-parasite levels of TbPCNA as a potential strategy for therapeutically exploiting this target in bloodstream form T. brucei.     

A phylogenetic study of two recently described endemic species of the Saxifraga granulata group from the central-north Mediterranean region (Italy) and their position in the context of the series Saxifraga (Saxifragaceae)

On two small islands of the Tuscan Archipelago, two new endemic species of the Saxifraga granulata group, namely S. caprariae and S. montis-christi, were recently described on a morphological basis. This led us to investigate these species using a molecular approach involving the analysis of ITS and trnL-F regions. Our aims were: to highlight their phylogenetic traits; to confirm or reject the hypothesis that they are more closely related to S. corsica than to S. granulata; and to verify their phylogenetic position in the context of Saxifraga L. ser. Saxifraga. We considered all the species belonging to the ser. Saxifraga and some other species of sect. Saxifraga – a total of 19 species. We generated 10 new DNA sequences, six for ITS and four for trnL-trnF. We produced separate Bayesian and Maximum likelihood trees for both ITS and trnL-trnF regions and we analysed the genetic distances among the new endemics and the more closely related species. The phylogenetic analyses showed that S. caprariae and S. montis-christi are relatively well separated from S. granulata and more related to S. corsica. This supports the taxonomic treatment previously proposed. We showed that the results of our phylogenetic analysis are in agreement with our biogeographic knowledge of the Tuscan Archipelago and the neighbouring territories and emphasize the conservation relevance of these findings.     

Glycosylations of Inosine and Uridine Nucleoside Bases and Synthesis of the New 1-(β-D-Glucopyranosyl)-Inosine-5′, 6″-diphosphate

Abstract

Gluco- and ribosylation of the bases of sugar protected inosine and uridine were investigated, obtaining only adducts with β-configuration at the new glycosidic carbon; stereospecific insertion of a sugar moiety at the 1-N of inosine was achieved either using a Mitsunobu approach (for ribosylation) or by direct coupling of 1-δ-bromoglucose 13 with 2′,3′,5′-tri-O-acetylinosine for glucosylation. 1-(β-D-glucosyl)-inosine, chosen as starting substrate for glucosylated analogs of cyclic IDP-ribose, was phosphorylated at the primary hydroxyls and tested in intramolecular pyrophosphate bond formation.     

Effects of the invasive duckweed Lemna minuta on aquatic animals: evidence from an indoor experiment

Abstract

Since effects of alien invasive free-floating plants can be relevant in aquatic ecosystems, we investigated the non-native Lemna minuta impact on four aquatic animal groups: Hydra vulgaris (Coelenterates), Asellus aquaticus (Arthropods), Gambusia affinis (Fish), Bufo bufo tadpoles (Amphibians). An indoor experiment was conducted keeping animals in water held in tanks with L. minuta mats of 0.5 (WI1), 1.5?cm thick (WI2) and without mats (WOU). Water parameters (DO, DO%, pH) and animal responses (survival rate, vitality) were measured every 48?h (0–288?h). Treatments with mats showed significant impacts on animals which were more severe with increasing mat thickness. Strong decreasing of oxygen and pH associated with mat occurrence had a large impact on animals. In WI2 all individuals died within 144?h (H. vulgaris, B. bufo within 96h), while in WI1 there was a higher survival rate and vitality (excluding B. bufo died within 96?h) and in WOU no deaths. This evidence suggests L. minuta thick mats (≥1.5?cm) could have a high impact on animal biodiversity, especially reducing oxygenation level in aquatic ecosystem.     

Adaptation of fungi,including yeasts,to cold environments

A wide range of cold environments exist, with an equally broad variety of fungi and yeasts that have adapted to such environments. These adaptations, which affect membranes, enzymes and other cellular components, such as radical scavenging molecules, display a great potential for exploitation in biotechnology. Alterations have been detected in membrane lipids, with an increase in fatty acid unsaturated bonds that enhance their fluidity. We report new data on the different phospholipid composition in membrane lipids in the same fungal species from both Antarctic and temperate regions. The decrease in temperature causes intracellular oxidative stress by inducing the generation of reactive oxygen species. We report the results of the first analysis of the non-enzymatic antioxidant response and phenolic compound production by an Antarctic strain of Geomyces pannorum. A survey on yeasts from the cryosphere is reported with a focus on their adaptation to a cold environment. Some studies have shown that the number of macrofungi in glacier forefronts rises as deglaciation increases. The survival success of many plants in such areas may be attributed to their mycorrhizal associations. We highlighted the macrofungal biodiversity of some Italian alpine habitats, in which we Inocybe microfastigiata, Laccaria montana and Lactarius salicis-herbaceae were recorded for the first time in Lombardy (Italy).     

The Entner–Doudoroff pathway empowers Pseudomonas putida KT2440 with a high tolerance to oxidative stress

Glucose catabolism of Pseudomonas putida is carried out exclusively through the Entner–Doudoroff (ED) pathway due to the absence of 6‐phosphofructokinase. In order to activate the Embden–Meyerhof–Parnas (EMP) route we transferred the pfkA gene from Escherichia coli to a P. putida wild‐type strain as well as to an eda mutant, i.e. lacking 2‐keto‐3‐deoxy‐6‐phosphogluconate aldolase. PfkA^{E. coli} failed to redirect the carbon flow from the ED route towards the EMP pathway, suggesting that ED was essential for sugar catabolism. The presence of PfkA^{E. coli} was detrimental for growth, which could be traced to the reduction of ATP and NAD(P)H pools along with alteration of the NAD(P)H/NADP⁺ ratio. Pseudomonas putida cells carrying PfkA^{E. coli} became highly sensitive to diamide and hydrogen peroxide, the response to which is very demanding of NADPH. The inhibitory effect of PfkA^{E. coli} could in part be relieved by methionine, the synthesis of which relies much on NADPH. These results expose the role of the ED pathway for generating the redox currency (NADPH) that is required for counteracting oxidative stress. It is thus likely that environmental bacteria that favour the ED pathway over the EMP pathway do so in order to gear their aerobic metabolism to endure oxidative‐related insults.