StaR Is a Positive Regulator of Topoisomerase I Activity Involved in Supercoiling Maintenance in Streptococcus pneumoniae

The DNA topoisomerases gyrase and topoisomerase I as well as the nucleoid-associated protein HU maintain supercoiling levels in Streptococcus pneumoniae, a main human pathogen. Here, we characterized, for the first time, a topoisomerase I regulator protein (StaR). In the presence of sub-inhibitory novobiocin concentrations, which inhibit gyrase activity, higher doubling times were observed in a strain lacking staR, and in two strains in which StaR was over-expressed either under the control of the ZnSO4-inducible PZn promoter (strain ΔstaRPZnstaR) or of the maltose-inducible PMal promoter (strain ΔstaRpLS1ROMstaR). These results suggest that StaR has a direct role in novobiocin susceptibility and that the StaR level needs to be maintained within a narrow range. Treatment of ΔstaRPZnstaR with inhibitory novobiocin concentrations resulted in a change of the negative DNA supercoiling density (σ) in vivo, which was higher in the absence of StaR (σ = -0.049) than when StaR was overproduced (σ = -0.045). We have located this protein in the nucleoid by using super-resolution confocal microscopy. Through in vitro activity assays, we demonstrated that StaR stimulates TopoI relaxation activity, while it has no effect on gyrase activity. Interaction between TopoI and StaR was detected both in vitro and in vivo by co-immunoprecipitation. No alteration of the transcriptome was associated with StaR amount variation. The results suggest that StaR is a new streptococcal nucleoid-associated protein that activates topoisomerase I activity by direct protein-protein interaction.This research and the APC were funded by project PID2021-124738OB-100 to A.G.d.l.C., financed by MCIN/AEI/10.13039/501100011033/FEDER, UE.S

Serum-Nutrient Starvation Induces Cell Death Mediated by Bax and Puma That Is Counteracted by p21 and Unmasked by Bcl-xL Inhibition

The cyclin-dependent kinase inhibitor p21 (p21WAF1/Cip1) is a multifunctional protein known to promote cell cycle arrest and survival in response to p53-dependent and p53 independent stimuli. We herein investigated whether and how it might contribute to the survival of cancer cells that are in low-nutrient conditions during tumour growth, by culturing isogenic human colorectal cancer cell lines (HCT116) and breast cancer cell lines in a medium deprived in amino acids and serum. We show that such starvation enhances, independently from p53, the expression of p21 and that of the pro-apoptotic BH3-only protein Puma. Under these conditions, p21 prevents Puma and its downstream effector Bax from triggering the mitochondrial apoptotic pathway. This anti-apoptotic effect is exerted from the cytosol but it is unrelated to the ability of p21 to interfere with the effector caspase 3. The survival function of p21 is, however, overcome by RNA interference mediated Bcl-xL depletion, or by the pharmacological inhibitor ABT-737. Thus, an insufficient supply in nutrients may not have an overt effect on cancer cell viability due to p21 induction, but it primes these cells to die, and sensitizes them to the deleterious effects of Bcl-xL inhibitors regardless of their p53 status

Three endo-β-mannanase genes expressed in the micropylar endosperm and in the radicle influence germination of Arabidopsis thaliana seeds

Mannans are hemicellulosic polysaccharides in the plant primary cell wall (CW). Mature seeds, specially their endosperm cells, have CWs rich in mannan-based polymers that confer a strong mechanical resistance for the radicle protrusion upon germination. The rupture of the seed coat and endosperm are two sequential events during the germination of Arabidopsis thaliana. Endo-β-mannanases (MAN; EC. 3.2.1.78) are hydrolytic enzymes that catalyze cleavage of β1 → 4 bonds in the mannan-polymer. In the genome of Arabidopsis, the endo-β-mannanase (MAN) family is represented by eight members. The expression of these eight MAN genes has been systematically explored in different organs of this plant and only four of them (AtMAN7, AtMAN6, AtMAN2 and AtMAN5) are expressed in the germinating seeds. Moreover, in situ hybridization analysis shows that their transcript accumulation is restricted to the micropylar endosperm and to the radicle and this expression disappears soon after radicle emergence. T-DNA insertion mutants in these genes (K.O. MAN7, K.O. MAN6, K.O. MAN5), except that corresponding to AtMAN2 (K.O. MAN2), germinate later than the wild type (Wt). K.O. MAN6 is the most affected in the germination time course with a t 50 almost double than that of the Wt. These data suggest that AtMAN7, AtMAN5 and specially AtMAN6 are important for the germination of A. thaliana seeds by facilitating the hydrolysis of the mannan-rich endosperm cell walls

The <i>Pratylenchus penetrans</i> transcriptome as a source for the development of alternative control strategies:mining for putative genes involved in parasitism and evaluation of <i>in planta</i> RNAi

The root lesion nematode Pratylenchus penetrans is considered one of the most economically important species within the genus. Host range studies have shown that nearly 400 plant species can be parasitized by this species. To obtain insight into the transcriptome of this migratory plant-parasitic nematode, we used Illumina mRNA sequencing analysis of a mixed population, as well as nematode reads detected in infected soybean roots 3 and 7 days after nematode infection. Over 140 million paired end reads were obtained for this species, and de novo assembly resulted in a total of 23,715 transcripts. Homology searches showed significant hit matches to 58% of the total number of transcripts using different protein and EST databases. In general, the transcriptome of P. penetrans follows common features reported for other root lesion nematode species. We also explored the efficacy of RNAi, delivered from the host, as a strategy to control P. penetrans, by targeted knock-down of selected nematode genes. Different comparisons were performed to identify putative nematode genes with a role in parasitism, resulting in the identification of transcripts with similarities to other nematode parasitism genes. Focusing on the predicted nematode secreted proteins found in this transcriptome, we observed specific members to be up-regulated at the early time points of infection. In the present study, we observed an enrichment of predicted secreted proteins along the early time points of parasitism by this species, with a significant number being pioneer candidate genes. A representative set of genes examined using RT-PCR confirms their expression during the host infection. The expression patterns of the different candidate genes raise the possibility that they might be involved in critical steps of P. penetrans parasitism. This analysis sheds light on the transcriptional changes that accompany plant infection by P. penetrans, and will aid in identifying potential gene targets for selection and use to design effective control strategies against root lesion nematodes

Demographic, clinical and antibody characteristics of patients with digital ulcers in systemic sclerosis: data from the DUO Registry

OBJECTIVES: The Digital Ulcers Outcome (DUO) Registry was designed to describe the clinical and antibody characteristics, disease course and outcomes of patients with digital ulcers associated with systemic sclerosis (SSc). METHODS: The DUO Registry is a European, prospective, multicentre, observational, registry of SSc patients with ongoing digital ulcer disease, irrespective of treatment regimen. Data collected included demographics, SSc duration, SSc subset, internal organ manifestations, autoantibodies, previous and ongoing interventions and complications related to digital ulcers. RESULTS: Up to 19 November 2010 a total of 2439 patients had enrolled into the registry. Most were classified as either limited cutaneous SSc (lcSSc; 52.2%) or diffuse cutaneous SSc (dcSSc; 36.9%). Digital ulcers developed earlier in patients with dcSSc compared with lcSSc. Almost all patients (95.7%) tested positive for antinuclear antibodies, 45.2% for anti-scleroderma-70 and 43.6% for anticentromere antibodies (ACA). The first digital ulcer in the anti-scleroderma-70-positive patient cohort occurred approximately 5 years earlier than the ACA-positive patient group. CONCLUSIONS: This study provides data from a large cohort of SSc patients with a history of digital ulcers. The early occurrence and high frequency of digital ulcer complications are especially seen in patients with dcSSc and/or anti-scleroderma-70 antibodies

Division I working group on "Precession and the ecliptic"

Abstract. The IAU Working Group on Precession and the Equinox looked at sev-eral solutions for replacing the precession part of the IAU 2000A precession–nutation model, which is not consistent with dynamical theory. These comparisons show that the (Capitaine et al., Astron. Astrophys., 412, 2003a) precession theory, P03, is both consis-tent with dynamical theory and the solution most compatible with the IAU 2000A nuta-tion model. Thus, the working group recommends the adoption of the P03 precession theory for use with the IAU 2000A nutation. The two greatest sources of uncertainty in the precession theory are the rate of change of the Earth’s dynamical flattening, J2, and the precession rates (i.e. the constants of integration used in deriving the precession). The combined uncertainties limit the accuracy in the precession theory to approximately 2mas cent−2. Given that there are difficulties with the traditional angles used to parameterize the precession, zA, ζA, and θA, the working group has decided that the choice of parame-ters should be left to the user. We provide a consistent set of parameters that may be used with either the traditional rotation matrix, or those rotation matrices described i