Anti-Virulence Potential of a Chionodracine-Derived Peptide against Multidrug-Resistant Pseudomonas aeruginosa Clinical Isolates from Cystic Fibrosis Patients

Pseudomonas aeruginosa is an opportunistic pathogen causing several chronic infections resistant to currently available antibiotics. Its pathogenicity is related to the production of different virulence factors such as biofilm and protease secretion. Pseudomonas communities can persist in biofilms that protect bacterial cells from antibiotics. Hence, there is a need for innovative approaches that are able to counteract these virulence factors, which play a pivotal role, especially in chronic infections. In this context, antimicrobial peptides are emerging drugs showing a broad spectrum of antibacterial activity. Here, we tested the anti-virulence activity of a chionodracine-derived peptide (KHS-Cnd) on five P. aeruginosa clinical isolates from cystic fibrosis patients. We demonstrated that KHS-Cnd impaired biofilm development and caused biofilm disaggregation without affecting bacterial viability in nearly all of the tested strains. Ultrastructural morphological analysis showed that the effect of KHS-Cnd on biofilm could be related to a different compactness of the matrix. KHS-Cnd was also able to reduce adhesion to pulmonary cell lines and to impair the invasion of host cells by P. aeruginosa. A cytotoxic effect of KHS-Cnd was observed only at the highest tested concentration. This study highlights the potential of KHS-Cnd as an anti-biofilm and anti-virulence molecule against P. aeruginosa clinical strains.     

Fine Mapping and Candidate Gene Analysis of Pm36, a Wild Emmer-Derived Powdery Mildew Resistance Locus in Durum Wheat

Powdery mildew (PM) is an economically important foliar disease of cultivated cereals worldwide. The cultivation of disease-resistant varieties is considered the most efficient, sustainable and economical strategy for disease management. The objectives of the current study were to fine map the chromosomal region harboring the wild emmer PM resistance locus Pm36 and to identify candidate genes by exploiting the improved tetraploid wheat genomic resources. A set of backcross inbred lines (BILs) of durum wheat were genotyped with the SNP 25K chip array and comparison of the PM-resistant and susceptible lines defined a 1.5 cM region (physical interval of 1.08 Mb) harboring Pm36. The genetic map constructed with F_2:3 progenies derived by crossing the PM resistant line 5BIL-42 and the durum parent Latino, restricted to 0.3 cM the genetic distance between Pm36 and the SNP marker IWB22904 (physical distance 0.515 Mb). The distribution of the marker interval including Pm36 in a tetraploid wheat collection indicated that the positive allele was largely present in the domesticated and wild emmer Triticum turgidum spp. dicoccum and ssp. dicoccoides. Ten high-confidence protein coding genes were identified in the Pm36 region of the emmer, durum and bread wheat reference genomes, while three added genes showed no homologous in the emmer genome. The tightly linked markers can be used for marker-assisted selection in wheat breeding programs, and as starting point for the Pm36 map-based cloning.     

Lightly supervised alignment of subtitles on multi-genre broadcasts

This paper describes a system for performing alignment of subtitles to audio on multigenre broadcasts using a lightly supervised approach. Accurate alignment of subtitles plays a substantial role in the daily work of media companies and currently still requires large human effort. Here, a comprehensive approach to performing this task in an automated way using lightly supervised alignment is proposed. The paper explores the different alternatives to speech segmentation, lightly supervised speech recognition and alignment of text streams. The proposed system uses lightly supervised decoding to improve the alignment accuracy by performing language model adaptation using the target subtitles. The system thus built achieves the third best reported result in the alignment of broadcast subtitles in the Multi–Genre Broadcast (MGB) challenge, with an F1 score of 88.8%. This system is available for research and other non–commercial purposes through webASR, the University of Sheffield’s cloud–based speech technology web service. Taking as inputs an audio file and untimed subtitles, webASR can produce timed subtitles in multiple formats, including TTML, WebVTT and SRT.     

Concomitant mobilization of plasma cells and hematopoietic progenitors into peripheral blood of multiple myeloma patients: positive selection and transplantation of enriched CD34+ cells to remove circulating tumor cells

One advantage of the use of peripheral blood stem cells (PBSCs) over autologous bone marrow would be a reduced risk of tumor cell contamination. However, the level of neoplastic cells in the PB of multiple myeloma (MM) patients after mobilization protocols is poorly investigated. In this study, we evaluated PB samples from 27 pretreated MM patients after the administration of high dose cyclophosphamide (7 g/m2 or 4 g/m2) and granulocyte-colony stimulating factor for the detection of myeloma cells as well as hematopoietic progenitors. Plasma cells containing intracytoplasmic lg were counted by microscope immunofluorescence after incubation with appropriate antisera directed against light- and heavy-chain lg. Moreover, flow cytometry studies were performed to determine the presence of malignant B-lineage elements by using monoclonal antibodies against the CD19 antigen and the monotypic light chain. Before initiation of PBSC mobilization, circulating plasma cells were detected in all MM patients in a percentage ranging from 0.1% to 1.8% of the mononuclear cell fraction (mean value, 0.7% +/- 0.4% SD). In these patients, a higher absolute number of PB neoplastic cells was detected after chemotherapy and granulocyte colony-stimulating factor. Kinetic analysis showed a pattern of tumor cell mobilization similar to that of normal hematopoietic progenitors with a maximum peak falling within the optimal time period for the collection of PBSCs. The absolute number of plasma cells showed a 10 to 50-fold increase as compared with the baseline value. Apheresis products contained 0.7% +/- 0.2% SD of myeloma cells (range, 0.2% to 2.7%). Twenty-three MM patients were submitted to PBSC collection. In 10 patients, circulating hematopoietic CD34+ cells were highly enriched by avidin-biotin immunoabsorption, were cryopreserved, and used to reconstitute bone marrow function after myeloablative therapy. The median purity of the enriched CD34+ cell population was 89.5% (range, 51% to 94%), with a 75-fold increase as compared with the pretreatment samples. The median overall recovery of CD34+ cells and colony-forming unit-granulocyte-macrophage was 58% (range, 33% to 95%) and 45% (range, 7% to 100%), respectively. Positive selection of CD34+ cells resulted in 2.5- to 3-log depletion of plasma cells and CD19+ B-lineage cells as determined by immunofluorescence studies, although DNA analysis of CDR III region of IgH gene showed the persistence of minimal residual disease in 5 of 6 patient samples studied. Myeloma patients were reinfused with enriched CD34+ cells after myeloablative therapy consisting of total body irradiation (1,000 cGy) and highdose melphalan (140 mg/m2). They received a median of 4 x 10(6) CD34+ cells/kg and showed a rapid reconstitution of hematopoiesis; the median time to 0.5 x 10(9) neutrophils and to 20 and 50 x 10(9) platelets per liter of PB was 10, 11, and 12 days, respectively. These results, as well as other clinically significant parameters, did not significantly differ from those of patients (n = 13) receiving unmanipulated PBSCs after the same pretransplant conditioning regimen. In summary, our data show the concomitant mobilization of tumor cells and hematopoietic progenitors in the PB of MM patients. Positive selection of CD34+ cells reduces the contamination of myeloma cells from the apheresis products up to 3-log and provides a cell suspension capable of restoring a normal hematopoiesis after a total body irradiation-containing conditioning regimen.     

Reptile heme protein structure: X-ray crystallographic study of the aquo-met and cyano-met derivatives of the loggerhead sea turtle (Caretta caretta) myoglobin at 2.0 A resolution

The X-ray crystal structures of the aquo-met and cyano-met derivatives of the loggerhead sea turtle (Caretta caretta) myoglobin have been determined at 2.0 A resolution (R = 0.182, and 0.178, respectively). The results here reported, representing the first reptile globin solved by X-ray crystallography, have been analyzed in parallel with data for related monomeric hemoproteins, and indicate a strong overall structural similarity between the loggerhead sea turtle and mammalian myoglobins, reflected by the 63% amino acid identity of their primary structures. The root-mean-square deviation between the entire polypeptide backbones of loggerhead sea turtle and sperm whale myoglobins, after structure superposition, is 0.83 A. Upon cyanide binding to the protein distal site, the iron-bound water molecule present in the aquo-met form is displaced by the incoming ligand. Cyanide is oriented towards the inner part of the heme distal site forming a Fe-C-N angle of 133 degrees.