Comparative study on the replication of HCV1b genome between wild‐type and cell culture‐adaptive mutant in regard to sensitivities against anti‐HCV drugs

2020 ◽  
Vol 64 (4) ◽  
pp. 296-303
Author(s):  
Yohei Miyayama ◽  
Heini Lee ◽  
HoJoong Song ◽  
Hiromi Abe‐Chayama ◽  
Daiki Miki ◽  
...  
Keyword(s):  
Cell Culture ◽  
Comparative Study ◽  
Wild Type

The URNA Genes of Herpesvirus Saimiri (Strain C488) Are Dispensable for Transformation of Human T Cells In Vitro

1999 ◽  
Vol 73 (12) ◽  
pp. 10551-10555 ◽  
Author(s):  
Armin Ensser ◽  
André Pfinder ◽  
Ingrid Müller-Fleckenstein ◽  
Bernhard Fleckenstein
Keyword(s):  
Cell Culture ◽  
Herpesvirus Saimiri ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Human T Cell ◽  
Human T Cells ◽  
Small Nuclear Rnas ◽  
T Cell Lines

ABSTRACT The herpesvirus saimiri strain C488 genome contains five genes for small nuclear RNAs, termed herpesvirus saimiri URNAs (or HSURs). Using a cosmid-based approach, all HSURs were precisely deleted from the genome. The mutant virus replicated at levels that were similar to those of wild-type viruses in OMK cells. Although the HSURs are expressed in wild-type virus-transformed human T-cell lines, the deletion does not affect viral transformation in cell culture.

scholarly journals The AML-associated K313 mutation enhances C/EBPα activity by leading to C/EBPα overexpression

2021 ◽  
Vol 12 (7) ◽  
Author(s):  
Ian Edward Gentle ◽  
Isabel Moelter ◽  
Mohamed Tarek Badr ◽  
Konstanze Döhner ◽  
Michael Lübbert ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Culture ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Transcriptional Activity ◽  
Target Gene ◽  
Wild Type ◽  
Elevated Expression ◽  
Factor C ◽  
Acute Myeloid

AbstractMutations in the transcription factor C/EBPα are found in ~10% of all acute myeloid leukaemia (AML) cases but the contribution of these mutations to leukemogenesis is incompletely understood. We here use a mouse model of granulocyte progenitors expressing conditionally active HoxB8 to assess the cell biological and molecular activity of C/EBPα-mutations associated with human AML. Both N-terminal truncation and C-terminal AML-associated mutations of C/EBPα substantially altered differentiation of progenitors into mature neutrophils in cell culture. Closer analysis of the C/EBPα-K313-duplication showed expansion and prolonged survival of mutant C/EBPα-expressing granulocytes following adoptive transfer into mice. C/EBPα-protein containing the K313-mutation further showed strongly enhanced transcriptional activity compared with the wild-type protein at certain promoters. Analysis of differentially regulated genes in cells overexpressing C/EBPα-K313 indicates a strong correlation with genes regulated by C/EBPα. Analysis of transcription factor enrichment in the differentially regulated genes indicated a strong reliance of SPI1/PU.1, suggesting that despite reduced DNA binding, C/EBPα-K313 is active in regulating target gene expression and acts largely through a network of other transcription factors. Strikingly, the K313 mutation caused strongly elevated expression of C/EBPα-protein, which could also be seen in primary K313 mutated AML blasts, explaining the enhanced C/EBPα activity in K313-expressing cells.

Time-resolved IR spectroscopy reveals mechanistic details of ion transport in the sodium pump Krokinobacter eikastus rhodopsin 2

2019 ◽  
Vol 21 (8) ◽  
pp. 4461-4471 ◽  
Author(s):  
Marvin Asido ◽  
Peter Eberhardt ◽  
Clara Nassrin Kriebel ◽  
Markus Braun ◽  
Clemens Glaubitz ◽  
...  
Keyword(s):  
Ir Spectroscopy ◽  
Comparative Study ◽  
Ion Transport ◽  
Structural Dynamics ◽  
Sodium Pump ◽  
Proton Pumping ◽  
Wild Type ◽  
Time Resolved

We report a comparative study on the structural dynamics of the light-driven sodium pump Krokinobacter eikastus rhodopsin 2 wild type under sodium and proton pumping conditions by means of time-resolved IR spectroscopy.

scholarly journals Rapid and Sensitive Direct Detection and Identification of Poliovirus from Stool and Environmental Surveillance Samples by Use of Nanopore Sequencing

2020 ◽  
Vol 58 (9) ◽  
Author(s):  
Alexander G. Shaw ◽  
Manasi Majumdar ◽  
Catherine Troman ◽  
Áine O’Toole ◽  
Blossom Benny ◽  
...  
Keyword(s):  
Cell Culture ◽  
Real Time ◽  
Environmental Samples ◽  
Direct Detection ◽  
Nanopore Sequencing ◽  
Wild Type ◽  
Consensus Sequences ◽  
Real Time Analysis ◽  
Detection And Identification ◽  
Culture Algorithm

ABSTRACT Global poliovirus surveillance involves virus isolation from stool and environmental samples, intratypic differential (ITD) by PCR, and sequencing of the VP1 region to distinguish vaccine (Sabin), vaccine-derived, and wild-type polioviruses and to ensure an appropriate response. This cell culture algorithm takes 2 to 3 weeks on average between sample receipt and sequencing. Direct detection of viral RNA using PCR allows faster detection but has traditionally faced challenges related to poor sensitivity and difficulties in sequencing common samples containing poliovirus and enterovirus mixtures. We present a nested PCR and nanopore sequencing protocol that allows rapid (<3 days) and sensitive direct detection and sequencing of polioviruses in stool and environmental samples. We developed barcoded primers and a real-time analysis platform that generate accurate VP1 consensus sequences from multiplexed samples. The sensitivity and specificity of our protocol compared with those of cell culture were 90.9% (95% confidence interval, 75.7% to 98.1%) and 99.2% (95.5% to 100.0%) for wild-type 1 poliovirus, 92.5% (79.6% to 98.4%) and 98.7% (95.4% to 99.8%) for vaccine and vaccine-derived serotype 2 poliovirus, and 88.3% (81.2% to 93.5%) and 93.2% (88.6% to 96.3%) for Sabin 1 and 3 poliovirus alone or in mixtures when tested on 155 stool samples in Pakistan. Variant analysis of sequencing reads also allowed the identification of polioviruses and enteroviruses in artificial mixtures and was able to distinguish complex mixtures of polioviruses in environmental samples. The median identity of consensus nanopore sequences with Sanger or Illumina sequences from the same samples was >99.9%. This novel method shows promise as a faster and safer alternative to cell culture for the detection and real-time sequencing of polioviruses in stool and environmental samples.

A Comparative Study on the Role of Cytokinins in Caryopsis Development in the Maizeminiature1Seed Mutant and Its Wild Type

2009 ◽  
Vol 51 (9) ◽  
pp. 840-849 ◽  
Author(s):  
Tomaž Rijavec ◽  
Maja Kovač ◽  
Aleš Kladnik ◽  
Prem S. Chourey ◽  
Marina Dermastia
Keyword(s):  
Comparative Study ◽  
Wild Type

scholarly journals Effect of Intragenic Rearrangement and Changes in the 3′ Consensus Sequence on NSP1 Expression and Rotavirus Replication

2001 ◽  
Vol 75 (5) ◽  
pp. 2076-2086 ◽  
Author(s):  
John T. Patton ◽  
Zenobia Taraporewala ◽  
Dayue Chen ◽  
Vladimir Chizhikov ◽  
Melinda Jones ◽  
...  
Keyword(s):  
Cell Culture ◽  
Consensus Sequence ◽  
Active Role ◽  
Serial Passage ◽  
Selective Advantage ◽  
Wild Type ◽  
Reading Frame ◽  
Group A Rotaviruses ◽  
Group A ◽  
Dsrna Genome

ABSTRACT The nonpolyadenylated mRNAs of rotavirus are templates for the synthesis of protein and the segmented double-stranded RNA (dsRNA) genome. During serial passage of simian SA11 rotaviruses in cell culture, two variants emerged with gene 5 dsRNAs containing large (1.1 and 0.5 kb) sequence duplications within the open reading frame (ORF) for NSP1. Due to the sequence rearrangements, both variants encoded only C-truncated forms of NSP1. Comparison of these and other variants encoding defective NSP1 with their corresponding wild-type viruses indicated that the inability to encode authentic NSP1 results in a small-plaque phenotype. Thus, although nonessential, NSP1 probably plays an active role in rotavirus replication in cell culture. In determining the sequences of the gene 5 dsRNAs of the SA11 variants and wild-type viruses, it was unexpectedly found that their 3′ termini ended with 5′-UGAACC-3′ instead of the 3′ consensus sequence 5′-UGACC-3′, which is present on the mRNAs of nearly all other group A rotaviruses. Cell-free assays indicated that the A insertion into the 3′ consensus sequence interfered with its ability to promote dsRNA synthesis and to function as a translation enhancer. The results provide evidence that the 3′ consensus sequence of the gene 5 dsRNAs of SA11 rotaviruses has undergone a mutation causing it to operate suboptimally in RNA replication and in the expression of NSP1 during the virus life cycle. Indeed, just as rotavirus variants which encode defective NSP1 appear to have a selective advantage over those encoding wild-type NSP1 in cell culture, it may be that the atypical 3′ end of SA11 gene 5 has been selected for because it promotes the expression of lower levels of NSP1 than the 3′ consensus sequence.

Immunolabeling of SSPE and wild-type measles viruses in ferret brain cell culture

1980 ◽  
Vol 50 (3) ◽  
pp. 181-186 ◽  
Author(s):  
Hannah R. Brown ◽  
Halldor Thormar ◽  
Fu Hai Lin
Keyword(s):  
Cell Culture ◽  
Brain Cell ◽  
Wild Type ◽  
Brain Cell Culture

scholarly journals SO026HUMAN KIDNEY PROXIMAL TUBULAR 3D SPHEROIDS TO STUDY THE ROLE OF ADAM17 IN DIABETIC NEPHROPATHY

2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
Vanesa Palau ◽  
Bramasta Nugraha ◽  
Maximilian Emmert ◽  
Simon Hoerstrup ◽  
Julio Pascual Santos ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Culture ◽  
Diabetic Nephropathy ◽  
High Glucose ◽  
Human Kidney ◽  
Collagen Iv ◽  
Control Group ◽  
Wild Type ◽  
Factor Α ◽  
3D Spheroids

Abstract Background and Aims ADAM17 is a disintegrin and metalloproteinase initially described to cleave the tumor necrosis factor α (TNFα). Currently, it is known that it can also release ectodomains of a diverse variety of molecules such as, transforming growth factor α (TGFα), L-selectin, and angiotensin-converting enzyme 2 (ACE2). It has been shown that ADAM17 protein expression increases in kidney mesangial cells after incubation with high glucose media mimicking what has been observed in diabetic patients and experimental models of diabetic nephropathy. We now studied the effect ADAM17 deletion on human kidney cells (HKC-8) in a 3D spheroids in vitro cell culture incubated with high glucose, low glucose and mannitol medium resembling the in vivo human kidney diabetic environment. Method ADAM17 deletion was performed using the CRISPR/Cas9 technology. HKC8 cells grew inside a RGD-functionalized dextran hydrogel to obtain 3D spheroids. 13 days post-seeding, the spheroids were incubated with 35mM of D-glucose (HG), 5mM of D-glucose (LG) or 35mM of mannitol as osmotic control for 6h, 24h or 72h. The quality of the established 3D cell culture of mature HKC-8 spheroids was assessed by Aquoporin-1 and Glut-1 staining. After incubations quantitative-PCR analyses were performed for fibrotic and inflammatory markers. Immunofluorescence for fibrotic markers was performed on HKC-8 spheroids incubated for 72h. Results High glucose (HG) medium induced CCL5 gene expression on wild-type HKC-8 spheroids after 6h and 24h of incubation in comparison with the control group. Interestingly, in the ADAM17-deleted spheroids, CCL5 gene expression maintained similar to control after 6h of incubation with HG medium and tended to decrease after 24h of incubation in comparison with the wild-type. Collagen IV gene expression was increased in the wild-type spheroids incubated with HG in comparison with the control group. In ADAM17-deleted spheroids, Collagen IV gene expression was significantly decreased in the cells incubated with HG in comparison with the wild-type cells incubated with HG. HG increased the expression of α-SMA, fibronectin and Collagen IV in wild-type spheroids. Adam17 deletion blocked the increase of α-SMA, fibronectin and Collagen IV expression compared with wild-type cells after 72h of incubation. Conclusion ADAM17 blockade protects against fibrosis and inflammation in human kidney tubular spheroids under high glucose.

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