Purification of genomic DNA by short monolithic columns Forcic, Dubravko; Branovic-Cakanic, Karmen; Ivancic, Jelena ...
Journal of Chromatography A,
02/2005, Letnik:
1065, Številka:
1
Journal Article, Conference Proceeding
Recenzirano
The isolation and purification of nucleic acids is essential for many procedures in molecular biology. After showing that bacterial and eukaryotic genomic DNA can be specifically bound to the CIM ...DEAE monolithic column, this characteristic was exploited in development of a simple and fast chromatographic procedure for isolation and purification of genomic DNA from cell lysates that does not include the usage of toxic organic solutions. The purity and the quality of the isolate as well as the duration of the procedure was similar to other chromatographic methods used today for isolation of genomic DNA, but the initial sample volume was not restricted.
Eleven mumps vaccine strains, all containing live attenuated virus, have been used throughout the world. Although L-Zagreb mumps vaccine has been licensed since 1972, only its partial nucleotide ...sequence was previously determined (accession numbers
AJ272363,
AY623666 and
AY583323). Therefore, we sequenced the entire genome of L-Zagreb vaccine strain (Institute of Immunology Inc., Zagreb, Croatia). In order to investigate the genetic stability of the vaccine, sequences of both L-Zagreb master seed and currently produced vaccine batch were determined and no difference between them was observed.
A phylogenetic analysis based on SH gene sequence has shown that L-Zagreb strain does not belong to any of established mumps genotypes and that it is most similar to old, laboratory preserved European strains (1950s–1970s).
L-Zagreb nucleotide and deduced protein sequences were compared with other mumps virus sequences obtained from the GenBank. Emphasis was put on functionally important protein regions and known antigenic epitopes. The extensive comparisons of nucleotide and deduced protein sequences between L-Zagreb vaccine strain and other previously determined mumps virus sequences have shown that while the functional regions of HN, V, and L proteins are well conserved among various mumps strains, there can be a substantial amino acid difference in antigenic epitopes of all proteins and in functional regions of F protein. No molecular pattern was identified that can be used as a distinction marker between virulent and attenuated strains.
Analysis of crude samples from biotechnological processes is often required to demonstrate that residual host cell impurities are reduced or eliminated during purification. Current knowledge suggests ...that a continuous-cell-line DNA can be considered a cellular contaminant rather than a significant risk factor requiring removal to extremely low levels. Anion-exchange chromatography is one of the most important methods used in the downstream processing and analysis of different biomolecules. In this article, an application using Convective Interaction Media monolithic columns to improve the detection of residual cellular DNA is described.
To confirm the genetic stability of the Edmonston–Zagreb vaccine strain, we determined and compared the nucleotide sequences of genuine Edmonston–Zagreb master seed (EZ D22) and current working seed ...lot (EZ D24 2/99). Sequence analysis and comparison of the two sequences confirmed that these two sequences are the same at the molecular level. The obtained sequences were also compared to reference strains, i.e. Edmonston wild-type (Edmonston Wt) AF266288 and Edmonston–Zagreb (EZ) AF266290 vaccine strain. The sequence of EZ D22 differed from the Edmonston Wt in 32 nucleotides. EZ D22 differed from EZ AF266290 in six nucleotides. Coding substitution at position 441 and two silent substitutions at positions 11999 and 14612 in the
L gene are unique to EZ D22. The differences found between EZ from different sources can be a good reason for periodical sequence analysis of the same strain in the hands of different manufactures.
Virus mumpsa uzročnik je zaušnjaka, bolesti koja se može prevenirati cijepljenjem. Mumps je RNK virus koji se u kliničkim uzorcima i u supernatantima inficiranih staničnih kultura nakon izolacije RNK ...može detektirati RT-PCR testom. Prednosti RT-PCR testa su brzina, specifičnost i mogućnost detekcije malog broja kopija virusa. Genskom karakterizacijom i genotipizacijom virusa omogućeno je epidemiološko praćenje distribucije i cirkuliranja divljih tipova virusa mumpsa koji uzrokuju epidemije i u procijepljenim populacijama. Molekularna detekcija i genska karakterizacija divljih i cjepnih virusa mumpsa provodi se u Republici Hrvatskoj od kraja 90-tih godina.
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•Interoperability among all integrated software (with some limitations explicitly mentioned in the paper).•The whole cryoET data processing pipeline is covered.•Modern visualization ...tools for cryoET data, like segmentation, oriented 3D coordinates, or a tilt series CTF analyzer.•Manages all the metadata and image file conversions.•Handles incompatibilities between different software packages.•Prevents users from scripting, losing track of the completed processing attempts, and using a logbook.
Image processing in cryogenic electron tomography (cryoET) is currently at a similar state as Single Particle Analysis (SPA) in cryogenic electron microscopy (cryoEM) was a few years ago. Its data processing workflows are far from being well defined and the user experience is still not smooth. Moreover, file formats of different software packages and their associated metadata are not standardized, mainly since different packages are developed by different groups, focusing on different steps of the data processing pipeline.
The Scipion framework, originally developed for SPA (de la Rosa-Trevín et al., 2016), has a generic python workflow engine that gives it the versatility to be extended to other fields, as demonstrated for model building (Martínez et al., 2020). In this article, we provide an extension of Scipion based on a set of tomography plugins (referred to as ScipionTomo hereafter), with a similar purpose: to allow users to be focused on the data processing and analysis instead of having to deal with multiple software installation issues and the inconvenience of switching from one to another, converting metadata files, managing possible incompatibilities, scripting (writing a simple program in a language that the computer must convert to machine language each time the program is run), etcetera. Additionally, having all the software available in an integrated platform allows comparing the results of different algorithms trying to solve the same problem. In this way, the commonalities and differences between estimated parameters shed light on which results can be more trusted than others. ScipionTomo is developed by a collaborative multidisciplinary team composed of Scipion team engineers, structural biologists, and in some cases, the developers whose software packages have been integrated. It is open to anyone in the field willing to contribute to this project.
The result is a framework extension that combines the acquired knowledge of Scipion developers in close collaboration with third-party developers, and the on-demand design of functionalities requested by beta testers applying this solution to actual biological problems.
Deregulated E2F transcription factor activity occurs in the vast majority of human tumors and has been solidly implicated in disturbances of cell cycle control, proliferation, and apoptosis. Aberrant ...E2F regulatory activity is often caused by impairment of control through pRB function, but little is known about the interplay of other oncoproteins with E2F. Here we show that ETS transcription factor fusions resulting from disease driving rearrangements in Ewing sarcoma (ES) and prostate cancer (PC) are one such class of oncoproteins. We performed an integrative study of genome-wide DNA-binding and transcription data in EWSR1/FLI1 expressing ES and TMPRSS2/ERG containing PC cells. Supported by promoter activity and mutation analyses, we demonstrate that a large fraction of E2F3 target genes are synergistically coregulated by these aberrant ETS proteins. We propose that the oncogenic effect of ETS fusion oncoproteins is in part mediated by the disruptive effect of the E2F-ETS interaction on cell cycle control. Additionally, a detailed analysis of the regulatory targets of the characteristic EWSR1/FLI1 fusion in ES identifies two functionally distinct gene sets. While synergistic regulation in concert with E2F in the promoter of target genes has a generally activating effect, EWSR1/FLI1 binding independent of E2F3 is predominantly associated with repressed differentiation genes. Thus, EWSR1/FLI1 appears to promote oncogenesis by simultaneously promoting cell proliferation and perturbing differentiation.
Primary hepatic neoplasms with neuroendocrine differentiation are extremely rare. Their clinicopathological features and molecular genetic basis are largely unknown. We identified four cases of ...primary hepatic neoplasms with neuroendocrine differentiation. Electronic medical records were reviewed for clinical history, imaging findings, laboratory results, and follow-up. Pathology slides, immunohistochemistry, and ancillary studies were reviewed. There were two females and two males with age ranging from 52 to 74 years. There was one amphicrine carcinoma with tumor cells simultaneously demonstrating both hepatocellular and neuroendocrine differentiation, one mixed hepatocellular-neuroendocrine carcinoma (NEC) with hepatocellular component intermingled with neuroendocrine component, one small cell NEC, and one well-differentiated neuroendocrine tumor. Next- generation sequencing of the mixed hepatocellular-NEC and small cell NEC showed molecular/genetic alterations commonly seen in hepatocellular carcinoma (HCC). All four cases arose in a background of cirrhosis. Primary hepatic neoplasms arising in cirrhotic livers can have a spectrum of neuroendocrine differentiation. Presence of a NEC component may be an indicator of aggressiveness. In addition, primary hepatic carcinomas with neuroendocrine differentiation likely share the same molecular pathways as HCC.
•Hepatic neoplasms in cirrhotic livers can have neuroendocrine differentiation.•Neuroendocrine carcinoma component may be an indicator of aggressiveness.•Hepatic neuroendocrine carcinoma likely share the same molecular pathways as HCC.
In the liver, ductal cells rarely proliferate during homeostasis but do so transiently after tissue injury. These cells can be expanded as organoids that recapitulate several of the cell-autonomous ...mechanisms of regeneration but lack the stromal interactions of the native tissue. Here, using organoid co-cultures that recapitulate the ductal-to-mesenchymal cell architecture of the portal tract, we demonstrate that a subpopulation of mouse periportal mesenchymal cells exerts dual control on proliferation of the epithelium. Ductal cell proliferation is either induced and sustained or, conversely, completely abolished, depending on the number of direct mesenchymal cell contacts, through a mechanism mediated, at least in part, by Notch signaling. Our findings expand the concept of the cellular niche in epithelial tissues, whereby not only soluble factors but also cell-cell contacts are the key regulatory cues involved in the control of cellular behaviors, suggesting a critical role for cell-cell contacts during regeneration.
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•The numbers of mesenchyme-ductal cell contacts change during damage regeneration•Ductal-mesenchyme organoids recapitulate cell interactions and in vivo architecture•Mesenchymal cells induce ductal epithelium cell proliferation via soluble factors•Mesenchymal cells impair ductal epithelium cell proliferation via cell-cell contacts
Huch and colleagues describe that during liver regeneration, ductal and mesenchymal cells proliferate with different tempos, resulting in changes in the cellular contacts between both populations. By developing organoid co-cultures that recapitulate ductal-mesenchymal interactions, they provide proof that the number of cell contacts matters and either promotes or arrests epithelial proliferation.
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