Paola Bernabò

Respiration rate and oxy-regulatory capacity in cold stenothermal chironomids.

The effects of temperature and oxygen saturation on the respiration rate of two cold stenothermal chironomids, Diamesa insignipes and Pseudodiamesa branickii were investigated. Fourth instar larvae were collected in winter in a glacio-rhithral stream (1300 m a.s.l., Alps, NE-Italy) and their respiration rate was measured with a Clarks electrode in the range 0-14 degrees C. The respiration rate was significantly higher in D. insignipes than in P. branickii at low temperatures (<or=4 degrees C), higher in P. branickii between 8 and 12 degrees C and comparable at 14 degrees C. Higher values of R (regulation value), R(25%) (respiration rate at 25% oxygen saturation) and b(1)/b(2) (slope ratio in piecewise linear regression), and lower values of P(c) (critical pressure) and I (initial decrease) were recorded in P. branickii than in D. insignipes. These values are compatible with oxy-regulatory behaviour in P. branickii, whereas D. insignipes appeared to be almost an oxy-conformer. On the basis of this autoecological information, new implications regarding survival of species from cold, high altitude habitats under changing climatic conditions are made.

Clinical populations of Pseudomonas aeruginosa isolated from acute infections show a wide virulence range partially correlated with population structure and virulence gene expression

Pseudomonas aeruginosa is a ubiquitous environmental bacterium responsible for a variety of infections in humans, as well as in animal hosts. While the evolution of virulence in P. aeruginosa strains isolated from chronic lung infection in cystic fibrosis (CF) patients has been extensively studied, the virulence phenotype of P. aeruginosa isolated from other infection types or from the environment is currently not well characterized. Here we report an extensive analysis of the virulence of P. aeruginosa strains isolated from acute infections compared with population structure. Virulence profiles of individual strains were also compared with the expression levels of the rhlR gene, the transcriptional regulator of the rhl quorum-sensing system, and the gene encoding Crc, a global regulator controlling catabolite repression and carbon metabolism. Additionally, the presence/absence of the two mutually exclusive genes, exoU and exoS, encoding effectors of the type III secretion system, was assessed. In order to capture the widest range of genetic variability, a collection of 120 clinical strains was initially characterized by repetitive element-based PCR genotyping, and a selection of 27 strains belonging to different clonal lineages was subsequently tested using three different virulence assays, including two Dictyostelium discoideum assays on different growth media, and a Caenorhabditis elegans fast-killing assay. We show that the parallel application of virulence assays can be used to quantitatively assess this complex, multifactorial phenotypic trait. We observed a wide spectrum of virulence phenotypes ranging from weakly to highly aggressive, indicating that clinical strains isolated from acute infections can present a reduced or altered virulence phenotype. Genotypic associations only partially correlated with virulence profiles and virulence gene expression, whereas the presence of either exoU or exoS was not significantly correlated with virulence. Interestingly, the expression of rhlR showed a significant and positive correlation with the virulence profiles obtained with the three assays, while the expression of crc was either negatively or not correlated with virulence, depending on the assay.

Three distinct ribosome assemblies modulated by translation are the building blocks of polysomes

Translation is increasingly recognized as a central control layer of gene expression in eukaryotic cells. The overall organization of mRNA and ribosomes within polysomes, as well as the possible role of this organization in translation are poorly understood. Here we show that polysomes are primarily formed by three distinct classes of ribosome assemblies. We observe that these assemblies can be connected by naked RNA regions of the transcript. We show that the relative proportions of the three classes of ribosome assemblies reflect, and probably dictate, the level of translational activity. These results reveal the existence of recurrent supra-ribosomal building blocks forming polysomes and suggest the presence of unexplored translational controls embedded in the polysome structure.

In Vivo Translatome Profiling in Spinal Muscular Atrophy Reveals a Role for SMN Protein in Ribosome Biology

Summary Genetic alterations impacting ubiquitously expressed proteins involved in RNA metabolism often result in neurodegenerative conditions, with increasing evidence suggesting that translation defects can contribute to disease. Spinal muscular atrophy (SMA) is a neuromuscular disease caused by low levels of SMN protein, whose role in pathogenesis remains unclear. Here, we identified in vivo and in vitro translation defects that are cell autonomous and SMN dependent. By determining in parallel the in vivo transcriptome and translatome in SMA mice, we observed a robust decrease in translation efficiency arising during early stages of disease. We provide a catalogue of RNAs with altered translation efficiency, identifying ribosome biology and translation as central processes affected by SMN depletion. This was further supported by a decrease in the number of ribosomes in SMA motor neurons in vivo. Overall, our findings suggest ribosome biology as an important, yet largely overlooked, factor in motor neuron degeneration.

Cold adaptive potential of chironomids overwintering in a glacial stream

Insects inhabiting cold streams must either tolerate or avoid freezing to survive. The present study reports the strategy adopted by fourth‐instar larvae of two chironomid species [Pseudodiamesa branickii (Nowicki) and Diamesa cinerella (Meigen)] overwintering in a glacial stream (in the Italian Alps). The cold adaptive potential of both species under acute cold stress is investigated down to –30 °C. Supercooling points, lower lethal temperatures (LLTs), haemolymph thermal hysteresis, whole body content of sugars and polyols, and the expression of heat shock protein (HSP) genes (hsc70 and hsp70) expression are estimated. Comparable thermal hysteresis (> 2 °C) is measured in the two species, both of which accumulate glucose and sucrose as the main cryoprotectants. According to the supercooling points (= –6.37 and –6.85 °C, respectively) and LLT100 (= –16.2 and –14.7 °C, respectively), P. branickii and D. cinerella can both be considered as freeze tolerant. However, the cumulative proportion of individual freezing values and the LLT50 (–9.14 and –6.13 °C, respectively) suggest that P. branickii is more cold hardy than D. cinerella, whereas the gene expression data (i.e. an absence of up‐regulation of hsp70 in D. cinerella) suggest that D. cinerella is more cold hardy than P. branickii. These findings are discussed in relation to the validity of the different metabolic indicators for defining the level of cold hardiness of a species, even in relation to its cold stenothermy. The results are also discussed in relation to climate warming, which represents a serious threat for species from glacier‐fed streams.

THERMAL STRESS INDUCES HSP70 PROTEINS SYNTHESIS IN LARVAE OF THE COLD STREAM NON-BITING MIDGE Diamesa cinerella Meigen

Laboratory experiments on the cold stenothermal midge Diamesa cinerella (Diptera, Chironomidae) were performed to study the relationship between increasing temperature and heat shock proteins (HSP70) expression at translational level (Western blotting). Thermotolerance of IV instar larvae collected in nature at 1.5-4.3°C during seasons was analyzed through short-term (1 h at ten different temperatures from 26°C to 35°C) and long-term (1-14 h at 26°C and 1-4 h at 32°C) heat shocks. A high thermotolerance was detected (LT50=30.9-32.8°C and LT100=34.0-37.8°C). However, survival decreased consistently with increasing exposure time, especially at higher temperature (LTime50=7.64 h at 26°C and LTime50=1.73 h at 32°C). The relationship between such heat resistance and HSP70 expression appeared evident because a relationship between HSP70 level and larval survival rate was generally found. A heat shock response (HSR) was consistent only in the summer larvae. The absence of HSR in the other populations coupled with even higher amounts of HSP70 than in summer, led us to hypothesize that other macromolecules and other adaptive mechanisms, apart from biochemical ones, are involved in the response of D. cinerella larvae to high temperature. Altogether these results stressed how in this midge the HSP70 protein family confers resistance against cold, being detected under natural conditions in control larvae collected in all seasons, but also against warm under experimental heat shocks. These results give new insights into possible responses to climate changes in freshwater insects within the context of global warming.

Transcriptional profiling induced by pesticides employed in organic agriculture in a wild population of Chironomus riparius under laboratory conditions

Copper (Cu) and azadirachtin (AZA-A+B) are pesticides allowed in organic agriculture whose environmental risk and toxicity for aquatic wildlife is only partially known. Reverse Transcription Polymerase Chain Reaction was used to assess the molecular effect of acute and short-term exposure (3, 24h) of Cu (0.01, 0.05, 1, 10, 25mgl(-1)) and AZA-A+B (0.2, 0.3, 0.4, 0.5, 1mgl(-1)) on the expression of five candidate genes (hsp70, hsc70, hsp40, hsp10 and cyP450) in a non-target species, Chironomus riparius. Fourth-instar larvae were collected from a mountain stream polluted by agricultural land run-off. All genes were responsive to both pesticides but each gene had a specific response to the different experimental concentrations and exposure times. A few similarities in transcriptional profiling were observed, such as a linear concentration-dependent response of hsp70 after 24h of exposure (at ≥1mgl(-1) of Cu and ≥0.2mgl(-1) of AZA-A+B) and an up-regulation regardless of the concentration of hsc70 after 24h of exposure (at ≥0mgl(-1) of Cu and ≥0.2mgl(-1) of AZA-A+B and the up-regulation of hsp70 after 3h of exposure at ~LC50 (Cu-LC50=26.1±2.5mgl(-1), AZA-A+B-LC50=1.1±0.2mgl(-1)). According to the results, hsp40, hsp10 and cyP450 may be defined as pesticide-dependent (i.e., hsp40 and hsp10 seemed to responded mainly to AZA-A+B and cyP450 to Cu), while hsc70 as time-dependent regardless of the pesticide (i.e., hsc70 responded only after 24h of treatment with Cu and AZA-A+B). This study gives new insights on the potential role of the C. ripariuss hsps and cyP450 genes as sensitive biomarkers for freshwater monitoring.

riboWaltz: Optimization of ribosome P-site positioning in ribosome profiling data

Ribosome profiling is a powerful technique used to study translation at the genome-wide level, generating unique information concerning ribosome positions along RNAs. Optimal localization of ribosomes requires the proper identification of the ribosome P-site in each ribosome protected fragment, a crucial step to determine the trinucleotide periodicity of translating ribosomes, and draw correct conclusions concerning where ribosomes are located. To determine the P-site within ribosome footprints at nucleotide resolution, the precise estimation of its offset with respect to the protected fragment is necessary. Here we present riboWaltz, an R package for calculation of optimal P-site offsets, diagnostic analysis and visual inspection of ribosome profiling data. Compared to existing tools, riboWaltz shows improved accuracies for P-site estimation and neat ribosome positioning in multiple case studies. riboWaltz was implemented in R and is available as an R package at https://github.com/LabTranslationalArchitectomics/RiboWaltz.

Peering at Brain Polysomes with Atomic Force Microscopy.

The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, formed by ribosomes, mRNAs, several proteins and non-coding RNAs, represent integrated platforms where translational controls take place. However, while the ribosome has been widely studied, the organization of polysomes is still lacking comprehensive understanding. Thus much effort is required in order to elucidate polysome organization and any novel mechanism of translational control that may be embedded. Atomic force microscopy (AFM) is a type of scanning probe microscopy that allows the acquisition of 3D images at nanoscale resolution. Compared to electron microscopy (EM) techniques, one of the main advantages of AFM is that it can acquire thousands of images both in air and in solution, enabling the sample to be maintained under near physiological conditions without any need for staining and fixing procedures. Here, a detailed protocol for the accurate purification of polysomes from mouse brain and their deposition on mica substrates is described. This protocol enables polysome imaging in air and liquid with AFM and their reconstruction as three-dimensional objects. Complementary to cryo-electron microscopy (cryo-EM), the proposed method can be conveniently used for systematically analyzing polysomes and studying their organization.

Thermal survival limits of young and mature larvae of a cold stenothermal chironomid from the Alps (Diamesinae: Pseudodiamesa branickii [Nowicki, 1873]).

The threats posed by climate change make it important to expand knowledge concerning cold and heat tolerance in stenothermal species from habitats potentially threatened by temperature changes. Thermal limits and basal metabolism variations were investigated in Pseudodiamesa branickii (Diptera: Chironomidae) under thermal stress between ‐20 and 37 °C. Supercooling point (SCP), lower (LLTs) and upper lethal temperatures (ULTs), and oxygen consumption rate were measured in overwintering young (1st and 2nd instar) and mature (3rd and 4th instar) larvae from an Alpine glacier‐fed stream. Both young and mature larvae were freezing tolerant (SCPs = ‐7.1 °C and ‐6.4 °C, respectively; LLT100 ‐20 °C) and thermotolerant (ULT50 = 31.7 ± 0.4, 32.5 ± 0.3, respectively). However, ontogenetic differences in acute tolerance were observed. The LLT50 calculated for the young larvae (= ‐7.4 °C) was almost equal to their SCP (= ‐7.1 °C) and the overlapping of the proportion of mortality curve with the CPIF curve highlighted that the young larvae are borderline between freezing tolerance and freezing avoidance. Furthermore, a lower ULT100 in the young larvae (of ca. 1 °C), suggests that they are less thermotolerant than mature larvae. Finally, young larvae exhibit a higher oxygen consumption rate (mgO2/gAFDM/h) at any temperature tested and are overall less resistant to oxygen depletion compared to mature larvae at ≥10 °C. These findings suggest that mature larvae enter into a dormant state by lowering their basal metabolism until environmental conditions improve in order to save energy for life cycle completion during stressful conditions.