Month: May 2020

Throughput sequencing setting, by combining VIDISCA with Roche 454 GS FLX Titanium sequencing. Nowadays molecular techniques are becoming the standard for the discovery of new viruses. Some methods use a conserved region for universal primer design, based on the known viral genomes. These methods are applicable to specific virus families, but cannot be used for all viruses. Furthermore, some yet unknown viruses could be too diverse and therefore remain negative in these kind of detection techniques. Sequence independent amplification methods, such as VIDISCA and random-PCR, can identify viral sequences without prior knowledge of a viral genome. Unfortunately, the detection of unknown viral pathogens in respiratory clinical material is difficult with these sequence independent virus discovery methods because of low viral load and high background nucleic acids in these samples. During the last years sequence independent virus discovery techniques were mostly used with virus BKM120 culture supernatant, as they contain high concentrations of viral genomes, or to discover previously unknown DNA viruses. So far no study has been able to identify novel human respiratory RNA viruses with sequence independent amplification techniques. Thus sequence independent amplification techniques like VIDISCA have to be optimized to allow discovery without requiring a culture amplification step. We managed to unfavor rRNA amplification by adjusting the reverse transcription step. Utilization of primers during cDNA synthesis that poorly recognize rRNA, in combination with the addition of oligo’s that halt cDNA synthesis on rRNA templates successfully decreased interfering background amplification. Additionally, using a single restriction enzyme with low numbers of recognition sites in 28S rRNA provided further reduction of useless and interfering amplification. Thus all steps increased the ratio of viral genome versus rRNA amplifications, and the benefit was shown in VIDISCA-high throughput sequencing of clinical samples containing known viruses. In the majority of clinical samples the virus was easily identified by VIDISCA-454. In two cases even an input of 140 and 190 genome copies of an adenovirus and influenza A virus could be detected by VIDISCA-454. Ideally, oldprotocol VIDISCA-454 should have been compared with optimized VIDISCA-454. However, this comparison is regrettably not possible due to limitation of the respiratory clinical specimens that we used. Thus we rely on all the reconstructions and monitoring performed with normal VIDISCA. As mentioned above, the use of one restriction enzyme diminished background rRNA amplification. There is one additional advantage of single restriction enzyme usage. In the traditional VIDISCA two restriction enzymes were combined and only fragments that have one restriction site on the 59 site and the other in the 39 site are amplified after ligation. Such VIDISCA amplification is restricted in case one of the two enzymes has few recognition sites optimal.

Carotid atherosclerosis has a high prevalence in adults with cardiovascular risk factors. Such carotid lesions have been shown to perturb baroreflex sensitivity as well as autonomic nervous system activity. Carotid atheroma induces locally structural changes that decrease the arterial distensibility with impairment of baroreflex sensitivity. The presence of carotid atheroma may also impair ventilatory flow characteristics through several pathways. The carotid plaque targets the carotid sinus, a structure known to be involved in the regulation of respiration. The baroreflex controls ventilation through changes in the frequency, tidal volume and breathing variability. Moreover, autonomic nervous system dysfunction occurs in the presence of a greater intima-media thickness of the carotid arteries and during carotid atherosclerosis. This sympatho-vagal imbalance could contribute to breathing control abnormalities as well. Consistent with our hypotheses, ventilatory chaos is SAR131675 VEGFR/PDGFR inhibitor impaired during carotid atherosclerosis. The main determinant of the flow dynamics alteration was the severity of the stenosis although other characteristics of the carotid plaque such as its localization and morphology also significantly decreased ventilatory chaos. Carotid endarterectomy reversed inspiratory flow chaos abnormalities, which reinforced the direct role played by the plaque in these alterations. Chaos impairment of inspiratory flow was strongly related to the severity of the carotid stenosis. In multiple regression analyses, the percentage of the carotid stenosis was the best in predicting the chaotic features of the inspiratory flow. However, other characteristics such as localization, surface features and morphology of the plaque also altered ventilatory chaos, independently of the degree of stenosis. The carotid plaques were classified depending on their echogenic features and surface morphology. The homogeneous echogenicity of the plaque is relied on histological characteristic such as collagen-rich, fibrous plaques, while heterogeneous echogenicity is found in lipid-rich or hemorrhagic plaques. Different local vascular consequences depending on the characteristics of the plaque may explain the differences evidenced in the largest Lyapunov exponent and the correlation dimension of the inspiratory flow. The structural changes of the vessel wall composition induce by the plaque directly impairs the baroreceptors and thus modifies the breathing pattern. In addition paracrine factors associated with atherosclerosis may potentiate the effects of structural modulation. Sympathovagal imbalance has also been described during carotid atheroma and this could contribute to ventilatory flow dynamics alterations. Indeed, baroreflex sensitivity has a significant effect in predicting the correlation dimension value and to a lesser extent the LLE of the inspiratory flow in multiple regression models.

Six species have been described, based on host preferences, metabolism, culture and antigenic features, including the two most recent species, isolated from seals and cetaceans, but most human cases remain to be caused by B. melitensis and B. abortus. However, DNA-DNA hybridization shows a high homology between strains, indicating that current species should be rather considered as subspecies corresponding to evolutionary lineages adapted to specific hosts. Classically, biphasic blood cultures such as the Ruiz-Castan˜eda method were used to isolate brucellae from blood and bone marrow. Now, most laboratories use continuous-monitoring automated blood culture systems, which can shorten the time to isolation and have been shown to be highly sensitive. Nevertheless, subculture is necessary to identify the microorganism, and brucellae may require 2–3 days to grow on chocolate or blood agar. Rapid automated bacterial identification systems must be interpreted with caution, because brucellae have been misidentified with some of these systems. PCR have shown high sensitivity and specificity, but its use CP-690550 side effects remains infrequent, mainly due to standardization problems. MALDI-TOF mass spectrometry has been suggested as a fast and reliable method for bacterial identification, based on protein profiles characteristic of each microorganism. Databases have been developed that include the main pathogenic microorganisms, thus allowing the use of this method in routine bacterial identification from plate culture. This is an important problem for the routine use of MALDI-TOF MS for the direct diagnosis of blood cultures in countries where brucellosis is still frequent. The aim of our study was to identify and differentiate Brucella species by MALDI-TOF MS, combining MALDI-TOF MS with dedicated bioinformatics and statistical methods. Initial spectra from three type strains of B. melitensis, five type strains of B. abortus and one type strain of B. suis, B. canis, B. ceti and B. pinnipedialis were used to set up database entries for re-identification of Brucella strains. This database was evaluated with 131 blind-coded Brucella clinical isolates previously identified by conventional methods. We also tested the reliability of this method for identifying brucellae directly from blood cultures. Brucellosis remains a serious problem in wide areas around the world. Symptoms are nonspecific. This lack of specificity of symptoms may delay the diagnosis for weeks. Though mortality rate is currently low, it remains a severe disease, and complications such as epididymoorchitis, arthritis, and CNS complications are not infrequent.

This coordination takes place in a “terminal knob” that is visible under electron microscopy which is a large 90S pre-ribosome complex known as ribosomal small subunit processome. This SSU processome contains 12S U3 snoRNP, MPP10 complex, t-UTPs, bUTP, BMS/RCL1 complex, RNA helicases and RNA-binding proteins. The coordination between Pol I transcription and rRNA processing is mediated by t-UTPs which are required for both 18S rRNA processing and Pol I transcription, and in addition, are associated with rDNA. Our previous study identified 1A6/DRIM as the human UTP20 which functions mainly in the processing of 18S rRNA. Proteomics studies have found that yeast UTP20 resides in the 90S preribosome in the nucleolus,. However, whether 1A6/DRIM is a classical UTP or a t-UTP remains unknown. In the present study, we investigated the function of 1A6/DRIM in Pol I transcription and found that 1A6/ DRIM functions as a t-UTP. Further study demonstrated that 1A6/DRIM affected UBF acetylation and raises the possibility that there is a novel mechanism by which t-UTPs activate Pol I transcription via altering modification of UBF or other Pol I transcription preinitiation complex factors. Pol I transcription and rRNA processing are coordinated and stringently controlled in normal cells. This coordination is mediated by the interaction of UBF with pre-rRNA processing factors. The SSU processome has been found to play a role in the coordination of Pol I transcription and pre-rRNA processing. The SSU processome is HhAntag691 assembled on nascent pre-rRNA as a large complex with a multimodular structure. While Pol I transcription is undergoing elongation, the SSU processome participates simultaneously in pre-rRNA processing. Pre-rRNA is sequentially cleaved at A0, A1 and A2. Upon cleavage at the A2 site, the pre-40S particle containing major components of the SSU processome and 18S rRNA departs from the remaining pre-rRNA on which another subset of ribosomal proteins are recruited to form the pre-60S particle. It is known that t-UTPs are required for Pol I transcription and early stages of pre-rRNA processing. Up to now, only seven t-UTPs have been identified in yeast and five t-UTPs have been identified in mammalian cells. However, the mechanisms by which t-UTPs function in Pol I transcription remain unknown. Ribosome biogenesis factors play crucial roles in cell proliferation, and deregulation of these factors, such as Pes1 which functions in 28S rRNA processing, often causes tumorigenesis,. Moreover, 1A6/DRIM has been found to be upregulated in some tumors and we are therefore currently investigating the 1A6/DRIM expression profile in human tumors to determine whether 1A6/DRIM can be used as a diagnostic marker or a therapeutic target in human tumors.

ES cells possess a unique cell cycle quite different from committed cells, especially G1 is shortened and Rb is hyperphosphorylated during the whole cell cycle, indicating that Ncl should not be influenced or in a complex with Rb in ES cells. Ncl is also described to interact with or be part of several transcription factor complexes, one of them being the B cellspecific transcription factor and switch region binding protein, LR1. Ncl has been shown to activate endogenous Bcl-2 and CD34 gene expression in CD34 positive hematopoietic cells by a direct sequence-specific interaction with the CD34 promoter region and is thus involved in the maintenance of these progenitor/stem cells in hematopoiesis. Even though Ncl is expressed at high levels in ES cells, not much is known about its specific role or physical interaction network in this cell type; therefore we explored Ncl in ES cells, starting with a search for new interaction partners. In current study, we show that Ncl interacts individually with translationally controlled tumor protein and Oct4 in a cell cycle dependent manner, and both Dasatinib complexes require phosphorylation of Ncl. The data presented here reveals new protein-protein complexes involving Ncl in murine and human ES cells, where we have identified Oct4 and Tpt1 as two endogenous Ncl-P interaction partners. All experiments with murine ES cells were performed on two different ES cell lines in parallel. We found no discernible differences between the ES cell lines, and one experiment also included human ES cells, indicating our findings to be generally applicable for ES cells, and not species or cell line specific. In our previous search for Tpt1 interaction partners in ES cells we found that Tpt1 forms a complex with Npm1. In the same screen additional potential interaction partners were found. One of the factors found were actin, which has recently been reported to interact with Tpt1 and we have now also revealed that NclP forms a complex with Tpt1 in murine ES cells. Ncl has previously been reported to be expressed at high levels in both murine and human ES cells where high levels of Tpt1 also were reported. Recent proteome analysis of mouse ES cell lines has furthermore revealed that the presence of Tpt1 is a characteristic of undifferentiated ES cells. Tpt1 is well conserved and expressed in all eukaryotic organisms but no main function has yet been found. Accumulating evidence demonstrate that Tpt1 and Ncl are important for cell proliferation. Tpt1 is up-regulated during the entry into the cell cycle. Over-expression of Tpt1 results in slow growing cells and a delayed cell cycle progression whereas over-expression of a Tpt1 double mutant, in which the two Plk1 phosphorylation sites have been substituted for alanines.