scholarly journals Continuous Cell Lines from the European Biting Midge Culicoides nubeculosus (Meigen, 1830)

2020 ◽  
Vol 8 (6) ◽  
pp. 825 ◽  
Author(s):  
Lesley Bell-Sakyi ◽  
Fauziah Mohd Jaafar ◽  
Baptiste Monsion ◽  
Lisa Luu ◽  
Eric Denison ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cultured Cells ◽  
Pcr Amplification ◽  
Laboratory Research ◽  
Biting Midges ◽  
Continuous Cell Lines ◽  
Transmission Electron ◽  
Western Palearctic ◽  
Serotype 1 ◽  
Culicoides Nubeculosus

Culicoides biting midges (Diptera: Ceratopogonidae) transmit arboviruses of veterinary or medical importance, including bluetongue virus (BTV) and Schmallenberg virus, as well as causing severe irritation to livestock and humans. Arthropod cell lines are essential laboratory research tools for the isolation and propagation of vector-borne pathogens and the investigation of host-vector-pathogen interactions. Here we report the establishment of two continuous cell lines, CNE/LULS44 and CNE/LULS47, from embryos of Culicoides nubeculosus, a midge distributed throughout the Western Palearctic region. Species origin of the cultured cells was confirmed by polymerase chain reaction (PCR) amplification and sequencing of a fragment of the cytochrome oxidase 1 gene, and the absence of bacterial contamination was confirmed by bacterial 16S rRNA PCR. Both lines have been successfully cryopreserved and resuscitated. The majority of cells examined in both lines had the expected diploid chromosome number of 2n = 6. Transmission electron microscopy of CNE/LULS44 cells revealed the presence of large mitochondria within cells of a diverse population, while arrays of virus-like particles were not seen. CNE/LULS44 cells supported replication of a strain of BTV serotype 1, but not of a strain of serotype 26 which is not known to be insect-transmitted. These new cell lines will expand the scope of research on Culicoides-borne pathogens.

Direct protein delivery into intact plant cells using polyhistidine peptides

2021 ◽  
Author(s):  
Yoshino Tanaka ◽  
Yoshihiko Nanasato ◽  
Kousei Omura ◽  
Keita Endoh ◽  
Tsuyoshi Kawano ◽  
...  
Keyword(s):  
Cell Lines ◽  
Fusion Proteins ◽  
Protein Delivery ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Plant Cells ◽  
Adenosine Monophosphate ◽  
Cell Penetrating Peptides ◽  
Intracellular Space ◽  
Intracellular Fluorescence

Abstract Polyhistidine peptides (PHPs), sequences comprising only histidine residues (>His8), are effective cell-penetrating peptides for plant cells. Using PHP-fusion proteins, we aimed to deliver proteins into cultured plant cells from Nicotiana tabacum, Oryza sativa, and Cryptomeria japonica. Co-cultivation of cultured cells with fusion proteins combining maltose-binding protein (MBP), red fluorescent protein (RFP), and various PHPs (MBP-RFP-His8–His20) in one polypeptide showed the cellular uptake of fusion proteins in all plant cell lines. Maximum intracellular fluorescence was shown in MBP-RFP-His20. Further, adenylate cyclase (CyaA), a synthase of cyclic adenosine monophosphate (cAMP) activated by cytosolic calmodulin, was used as a reporter for protein delivery in living cells. A fusion protein combining MBP, RFP, CyaA, and His20 (MBP-RFP-CyaA-His20) was delivered into plant cells and increased intracellular fluorescence and cAMP production in all cell lines. The present study demonstrates that PHPs are effective carriers of proteins into the intracellular space of various cultured plant cells.

scholarly journals Structure-guided selection of puromycin N-acetyltransferase mutants with enhanced selection stringency for deriving mammalian cell lines expressing recombinant proteins

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Alessandro T. Caputo ◽  
Oliver M. Eder ◽  
Hana Bereznakova ◽  
Heleen Pothuis ◽  
Albert Ardevol ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mammalian Cell ◽  
Recombinant Proteins ◽  
Cultured Cells ◽  
Hek293 Cells ◽  
Reaction Products ◽  
Enzyme Form ◽  
X Ray Crystallography ◽  
Mammalian Cell Lines ◽  
Apparent Loss

AbstractPuromycin and the Streptomyces alboniger-derived puromycin N-acetyltransferase (PAC) enzyme form a commonly used system for selecting stably transfected cultured cells. The crystal structure of PAC has been solved using X-ray crystallography, revealing it to be a member of the GCN5-related N-acetyltransferase (GNAT) family of acetyltransferases. Based on structures in complex with acetyl-CoA or the reaction products CoA and acetylated puromycin, four classes of mutations in and around the catalytic site were designed and tested for activity. Single-residue mutations were identified that displayed a range of enzymatic activities, from complete ablation to enhanced activity relative to wild-type (WT) PAC. Cell pools of stably transfected HEK293 cells derived using two PAC mutants with attenuated activity, Y30F and A142D, were found to secrete up to three-fold higher levels of a soluble, recombinant target protein than corresponding pools derived with the WT enzyme. A third mutant, Y171F, appeared to stabilise the intracellular turnover of PAC, resulting in an apparent loss of selection stringency. Our results indicate that the structure-guided manipulation of PAC function can be utilised to enhance selection stringency for the derivation of mammalian cell lines secreting elevated levels of recombinant proteins.

scholarly journals Susceptibility of Midge and Mosquito Vectors to SARS-CoV-2

2021 ◽  
Author(s):  
Velmurugan Balaraman ◽  
Barbara S Drolet ◽  
Natasha N Gaudreault ◽  
William C Wilson ◽  
Jeana Owens ◽  
...  
Keyword(s):  
Cell Lines ◽  
Potential Role ◽  
Animal Origin ◽  
Culex Tarsalis ◽  
Biting Midges ◽  
Bacterial Diseases ◽  
Culicoides Sonorensis ◽  
Insect Cell Lines ◽  
Intrathoracic Inoculation

Abstract SARS-CoV-2 is a recently emerged, highly contagious virus and the cause of the current COVID-19 pandemic. It is a zoonotic virus, although its animal origin is not clear yet. Person-to-person transmission occurs by inhalation of infected droplets and aerosols, or by direct contact with contaminated fomites. Arthropods transmit numerous viral, parasitic, and bacterial diseases; however, the potential role of arthropods in SARS-CoV-2 transmission is not fully understood. Thus far, a few studies have demonstrated that SARS-CoV-2 replication is not supported in cells from certain insect species nor in certain species of mosquitoes after intrathoracic inoculation. In this study, we expanded the work of SARS-CoV-2 susceptibility to biting insects after ingesting a SARS-CoV-2-infected bloodmeal. Species tested included Culicoides sonorensis (Wirth & Jones) (Diptera: Ceratopogonidae) biting midges, as well as Culex tarsalis (Coquillett) and Culex quinquefasciatus (Say) mosquitoes (Diptera: Culicidae), all known biological vectors for numerous RNA viruses. Arthropods were allowed to feed on SARS-CoV-2-spiked blood and at a time point postinfection analyzed for the presence of viral RNA and infectious virus. Additionally, cell lines derived from C. sonorensis (W8a), Aedes aegypti (Linnaeus) (Diptera: Culicidae) (C6/36), Cx. quinquefasciatus (HSU), and Cx. tarsalis (CxTrR2) were tested for SARS-CoV-2 susceptibility. Our results indicate that none of the biting insects, nor the insect cell lines evaluated support SARS-CoV-2 replication, suggesting that these species are unable to be biological vectors of SARS-CoV-2.

Isolation of Arboviruses From Cattle and Insects at 2 Sentinel Sites in Queensland, Australia, 1979-85

1990 ◽  
Vol 38 (1) ◽  
pp. 25 ◽  
Author(s):  
DH Cybinski ◽  
MJ Muller
Keyword(s):  
Cell Cultures ◽  
Virus Isolation ◽  
Hemorrhagic Disease ◽  
Tissue Cultures ◽  
Akabane Virus ◽  
Biting Midges ◽  
Bovine Ephemeral Fever ◽  
Serotype 1 ◽  
Intrathoracic Inoculation ◽  
First Time

Blood samples were collected regularly from two sentinel herds of cattle in northern and southern Queensland between 1979 and 1985. From 2660 samples, virus isolation attempts using baby hamster kidney (BHK21) and Aedes albopictus (AA) tissue cultures and suckling mice produced 308 viruses of which 243 (79%) were in the Palyam subgroup of orbiviruses. Mosquitoes and biting midges were collected at the southern sentinel herd site in January-February 1984 and processed for virus isolation in BHK2l and AA tissue cultures and by intrathoracic inoculation of Ae. aegypti mosquitoes. Totals of 14 338 midges of four species in 156 pools, and 9030 mosquitoes of 27 species in 232 pools, were processed and yielded 59 isolations. Of the 35 viruses isolated from Culicoides brevitarsis, 17 were members of the Palyam subgroup. Bovine ephemeral fever (BEF) virus was isolated once from Anopheles bancroftii, once from C. brevitarsis and 17 times from cattle. Akabane virus was isolated for the first time from C. wadai, as well as a further 10 times from C. brevitarsis and 20 times from cattle. Other viruses isolated from cattle included bluetongue serotype 1, and serotypes 5, 6 and 7 of epizootic hemorrhagic disease of deer (EHD). A new BEF group virus, tentatively called Oak Vale, was isolated nine times from Culex edwardsi mosquitoes. Of the orbiviruses, those in the Palyam subgroup were isolated almost exclusively in BHK2l tissue cultures but those in the bluetongue and EHD subgroups were isolated almost exclusively in AA cell cultures or after passage through Ae. aegypti. Of 22 rhabdovirus isolations from blood and insects (BEF, Kimberley and Tibrogargan), 16 were made only in AA cell cultures or after passage through Ae. aegypti.

scholarly journals Induction of Multiple Prophages from a Marine Bacterium: a Genomic Approach

2006 ◽  
Vol 72 (7) ◽  
pp. 4995-5001 ◽  
Author(s):  
Feng Chen ◽  
Kui Wang ◽  
Jeneen Stewart ◽  
Robert Belas
Keyword(s):  
Mitomycin C ◽  
Marine Bacterium ◽  
Pcr Amplification ◽  
Pulsed Field Gel Electrophoresis ◽  
Bacterial Genomes ◽  
Dominant Type ◽  
Transmission Electron ◽  
Viral Particles ◽  
Genomic Approach ◽  
Primer Sets

ABSTRACT Approximately 70% of sequenced bacterial genomes contain prophage-like structures, yet little effort has been made to use this information to determine the functions of these elements. The recent genomic sequencing of the marine bacterium Silicibacter sp. strain TM1040 revealed five prophage-like elements in its genome. The genomes of these prophages (named prophages 1 to 5) are approximately 74, 30, 39, 36, and 15 kb long, respectively. To understand the function of these prophages, cultures of TM1040 were treated with mitomycin C to induce the production of viral particles. A significant increase in viral counts and a decrease in bacterial counts when treated with mitomycin C suggested that prophages were induced from TM1040. Transmission electron microscopy revealed one dominant type of siphovirus, while pulsed-field gel electrophoresis demonstrated two major DNA bands, equivalent to 35 and 75 kb, in the lysate. PCR amplification with primer sets specific to each prophage detected the presence of prophages 1, 3, and 4 in the viral lysate, suggesting that these prophages are inducible, but not necessarily to the same level, while prophages 2 and 5 are likely defective or non-mitomycin C-inducible phages. The combination of traditional phage assays and modern microbial genomics provides a quick and efficient way to investigate the functions and inducibility of prophages, particularly for a host harboring multiple prophages with similar sizes and morphological features.

Effect of Granulocytic Chalone on the Growth Rate of Continuous Cell Lines Propagated in Vitro A New Assay System

2009 ◽  
Vol 29 (3) ◽  
pp. 257-264 ◽  
Author(s):  
P. Foa ◽  
A. T. Maiolo ◽  
L. Lombardi ◽  
T. Rytomaa ◽  
E. E. Polli
Keyword(s):  
Growth Rate ◽  
Cell Lines ◽  
Assay System ◽  
Continuous Cell Lines

scholarly journals Establishment of Cell Lines from Both Myeloma Bone Marrow and Plasmacytoma: SNU_MM1393_BM and SNU_MM1393_SC from a Single Patient

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Youngil Koh ◽  
Woo-June Jung ◽  
Kwang-Sung Ahn ◽  
Sung-Soo Yoon
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Lines ◽  
Mononuclear Cells ◽  
Cultured Cells ◽  
Clonal Evolution ◽  
Myeloma Cell ◽  
Mouse Bone Marrow ◽  
Single Patient ◽  
U266 Cell

Purpose.We tried to establish clinically relevant human myeloma cell lines that can contribute to the understanding of multiple myeloma (MM).Materials and Methods.Mononuclear cells obtained from MM patient’s bone marrow were injected via tail vein in an NRG/SCID mouse. Fourteen weeks after the injection, tumor developed at subcutis of the mouse. The engraftment of MM cells into mouse bone marrow (BM) was also observed. We separated and cultured cells from subcutis and BM.Results.After the separation and culture of cells from subcutis and BM, we established two cell lines originating from a single patient (SNU_MM1393_BM and SNU_MM1393_SC). Karyotype of the two newly established MM cell lines showed tetraploidy which is different from the karyotype of the patient (diploidy) indicating clonal evolution. In contrast to SNU_MM1393_BM, cell proliferation of SNU_MM1393_SC was IL-6 independent. SNU_MM1393_BM and SNU_MM1393_SC showed high degree of resistance against bortezomib compared to U266 cell line. SNU_MM1393_BM had the greater lethality compared to SNU_MM1393_SC.Conclusion.Two cell lines harboring different site tropisms established from a single patient showed differences in cytokine response and lethality. Our newly established cell lines could be used as a tool to understand the biology of multiple myeloma.

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