Site-directed mutagenesis of conserved inverted repeat sequences in the xylanase C promoter region from Streptomyces sp EC3

Streptomyces sp. EC3, a strain which was originally isolated from cattle manure compost, was shown to possess a strong xylanolytic activity. One of the genes responsible for this activity, xlnC, encodes a secreted xylanase. In the native strain, as in the heterologous host S. lividans, expression of xlnC was detectable in the presence of xylan but not in the presence of glucose. Induction by xylan was shown to take place at the transcriptional level. The transcriptional start site of xlnC was mapped and likely -35 (5'-TTGACA-3') and -10 (5'-GAGAAC-3') motifs were identified. In order to localise putative conserved regulatory sequences, the promoter regions of xylanase-encoding genes from various Streptomyces species were aligned. This alignment revealed the existence of three sets of quite well conserved palindromic AT rich sequences called boxes 1, 2 and 3. Box 3 (5'-CGAAA N TTTCG-3') is the farthest away from the promoter region (150-200 bp). A shorter version of this palindrome (5'-GAAA NN TTTC-3') or (5'-CGAAA-3') constitutes box 1, which is located just upstream of the putative -35 promoter sequence. Box 2, located 5-7 bp upstream of box 1, comprises a shorter palindrome than box 3, with inverted polarity [5'-(G/C)TTTC (N) GAAA(G/C)-3']. The putative regulatory role of the conserved inverted repeats in boxes 2 and 3 in the promoter region of the xlnC gene from Streptomyces sp. EC3, was assessed. These boxes were modified by site-directed mutagenesis, and the mutant promoter regions, as well as the wild-type promoter region, were separately fused to a beta-lactamase reporter gene. Analysis of the expression patterns of these fusions in cultures grown in the presence of glucose, xylan or both carbon sources demonstrated that these motifs were cis -acting negative regulatory elements, each playing a specific role in the regulation of xlnC expression. Box 3 was shown to be critical for the establishment of repression of xlnC expression by glucose, whereas box 2 was shown to play an important role in the induction of xlnC expression by xylan.Peer reviewe

Frequency-Dependent Reduction of Cybersickness in Virtual Reality by Transcranial Oscillatory Stimulation of the Vestibular Cortex

Virtual reality (VR) applications are pervasive of everyday life, as in working, medical, and entertainment scenarios. There is yet no solution to cybersickness (CS), a disabling vestibular syndrome with nausea, dizziness, and general discomfort that most of VR users undergo, which results from an integration mismatch among visual, proprioceptive, and vestibular information. In a double-blind, controlled trial, we propose an innovative treatment for CS, consisting of online oscillatory imperceptible neuromodulation with transcranial alternating current stimulation (tACS) at 10 Hz, biophysically modelled to reach the vestibular cortex bilaterally. tACS significantly reduced CS nausea in 37 healthy subjects during a VR rollercoaster experience. The effect was frequency-dependent and placebo-insensitive. Subjective benefits were paralleled by galvanic skin response modulation in 25 subjects, addressing neurovegetative activity. Besides confirming the role of transcranially delivered oscillations in physiologically tuning the vestibular system function (and dysfunction), results open a new way to facilitate the use of VR in different scenarios and possibly to help treating also other vestibular dysfunctions

Geomonumental routes: the granitic bridges over the Guadarrama river (Madrid, Spain) and the calcarenitic coastal towers from the Salento (Italy)

8 pages, 7 figures.-- Published in: Proceedings of the 11th International Congress on Deterioration and Conservation of Stone (Torun Poland, 15-20 September 2008), eds. Jadwiga W. Lukaszewicz and Piotr Niemcewicz.-- Presentation in PDF format available at: https://digital.csic.es/handle/10261/7648.This paper focuses on the new concept of GeoMonumental Routes, which mainly consists of the dissemination of architectural heritage with the added value of geology. Geology, so far, has not been considered in all its aspects in architectural heritage: i.e. geography, geomorphology, quarries provenance, building stones, and their relationship with historical and architectural aspects, constructive techniques and technological developments, as well as the connection of heritage structures to the settlement of historical routes. For this purpose, two scientific teams have gathered to develop two of this kind of routes following a common methodology, based on different geographical context, geological settlement, history, structure tipology and building stones.Thanks are given to both Ministries of Education from Spain and Italy for granting the CSIC-CNR bilateral cooperation project (2006IT0021).Peer reviewe

Endothelial cell-derived nidogen-1 inhibits migration of SK-BR-3 breast cancer cells

BACKGROUND: The tumour microenvironment is a critical regulator of malignant cancer progression. While endothelial cells have been widely studied in the context of tumour angiogenesis, their role as modulators of cancer cell invasion and migration is poorly understood. METHODS: We have investigated the influence of endothelial cells on the invasive and migratory behaviour of human cancer cells in vitro. RESULTS: Upon exposure to culture supernatants of endothelial cells, distinct cancer cells, such as SK-BR-3 cells, showed significantly increased invasion and cell migration concomitant with changes in cell morphology and gene expression reminiscent of an epithelial-mesenchymal transition (EMT). Interestingly, the pro-migratory effect on SK-BR-3 cells was significantly enhanced by supernatants obtained from subconfluent, proliferative endothelial cells rather than from confluent, quiescent endothelial cells. Systematically comparing the supernatants of subconfluent and confluent endothelial cells by quantitative MS proteomics revealed eight candidate proteins that were secreted at significantly higher levels by confluent endothelial cells representing potential inhibitors of cancer cell migration. Among these proteins, nidogen-1 was exclusively expressed in confluent endothelial cells and was found to be necessary and sufficient for the inhibition of SK-BR-3 cell migration. Indeed, SK-BR-3 cells exposed to nidogen-1-depleted endothelial supernatants showed increased promigratory STAT3 phosphorylation along with increased cell migration. This reflects the situation of enhanced SK-BR-3 migration upon stimulation with conditioned medium from subconfluent endothelial cells with inherent absence of nidogen-1 expression. CONCLUSION: The identification of nidogen-1 as an endothelial-derived inhibitor of migration of distinct cancer cell types reveals a novel mechanism of endothelial control over cancer progression

Anti-ganglioside antibodies : experience from the Italian Association of Neuroimmunology external quality assessment scheme

Anti-ganglioside antibodies are currently used in the differential diagnosis of suspected immune-mediated neuropathies. In-house and increasingly used commercial assays seem to perform suboptimally, and comparative information on their analytical performance are essentially lacking. Born within the frame of guidelines and standardization activities by the Italian Association of Neuroimmunology, this external quality assessment scheme (EQAS) is a real-life snapshot of the laboratory diagnostics in this field. The EQAS consisted of five surplus, anonymized serum samples from patients with clinically-defined neuropathies and two serum samples from healthy blood donors. Eight laboratories used commercial line-/dot-blots, seven in-house/commercial ELISAs (in addition, 13 laboratories tested a recently released ELISA by B\ufchlmann). Only high anti-ganglioside antibody reactivities were considered, in accordance with consolidated recommendations. Large variations in anti-ganglioside antibody profiles were observed, even, although to a lesser extent, within homogeneous classes of assays. Concordance between the profiles and clinical phenotypes was also partial. Although conducted on a relatively small, but representative number of Italian laboratories, this EQAS shows a critical between-laboratory disagreement in the test results of anti-ganglioside antibodies. Also considering the trend for using certified assays in generalist laboratories, strong efforts toward standardization and the identification of the best method(s) for their determinations are compellingly needed

Breakthrough SARS-CoV-2 infections in MS patients on disease-modifying therapies

Background: Patients with multiple sclerosis (pwMS) treated with anti-CD20 or fingolimod showed a reduced humoral response to SARS-CoV-2 vaccines. Objective: In this study we aimed to monitor the risk of breakthrough SARS-CoV-2 infection in pwMS on different disease-modifying therapies (DMTs). Methods: Data on the number of vaccinated patients and the number of patients with a breakthrough infection were retrospectively collected in 27 Italian MS centers. We estimated the rate of breakthrough infections and of infection requiring hospitalization per DMT. Results: 19,641 vaccinated pwMS were included in the database. After a median follow-up of 8 months, we observed 137 breakthrough infections. Compared with other DMTs, the rate of breakthrough infections was significantly higher on ocrelizumab (0.57% vs 2.00%, risk ratio (RR) = 3.55, 95% CI = 2.74-4.58, p < 0.001) and fingolimod (0.58% vs 1.62%, RR = 2.65, 95% CI = 1.75-4.00, p < 0.001), while there were no significant differences in any other DMT group. In the ocrelizumab group the hospitalization rate was 16.7% versus 19.4% in the pre-vaccination era (RR = 0.86, p = 0.74) and it was 3.9% in all the other DMT groups versus 11.9% in the pre-vaccination period (RR = 0.33, p = 0.02). Conclusions: The risk of breakthrough SARS-CoV-2 infections is higher in patients treated with ocrelizumab and fingolimod, and the rate of severe infections was significantly reduced in all the DMTs excluding ocrelizumab