scholarly journals Enhancement of Campylobacter hepaticus culturing to facilitate downstream applications

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Canh Phung ◽  
Timothy B. Wilson ◽  
José A. Quinteros ◽  
Peter C. Scott ◽  
Robert J. Moore ◽  
...  
Keyword(s):  
Liquid Culture ◽  
Vaccine Development ◽  
Genomic Analysis ◽  
Bacterial Biomass ◽  
In Silico Analysis ◽  
Culture Method ◽  
Efficient Production ◽  
Culture Density ◽  
Brucella Broth

AbstractCampylobacter hepaticus causes Spotty Liver Disease (SLD) in chickens. C. hepaticus is fastidious and slow-growing, presenting difficulties when growing this bacterium for the preparation of bacterin vaccines and experimental disease challenge trials. This study applied genomic analysis and in vitro experiments to develop an enhanced C. hepaticus liquid culture method. In silico analysis of the anabolic pathways encoded by C. hepaticus revealed that the bacterium is unable to biosynthesise l-cysteine, l-lysine and l-arginine. It was found that l-cysteine added to Brucella broth, significantly enhanced the growth of C. hepaticus, but l-lysine or l-arginine addition did not enhance growth. Brucella broth supplemented with l-cysteine (0.4 mM), l-glutamine (4 mM), and sodium pyruvate (10 mM) gave high-density growth of C. hepaticus and resulted in an almost tenfold increase in culture density compared to the growth in Brucella broth alone (log10 = 9.3 vs 8.4 CFU/mL). The type of culture flask used also significantly affected C. hepaticus culture density. An SLD challenge trial demonstrated that C. hepaticus grown in the enhanced culture conditions retained full virulence. The enhanced liquid culture method developed in this study enables the efficient production of bacterial biomass and therefore facilitates further studies of SLD biology and vaccine development.

scholarly journals A Rapid Method for Quantifying Viable Mycobacterium avium subsp. paratuberculosis in Cellular Infection Assays

2016 ◽  
Vol 82 (18) ◽  
pp. 5553-5562 ◽  
Author(s):  
Hannah B. Pooley ◽  
Kumudika de Silva ◽  
Auriol C. Purdie ◽  
Douglas J. Begg ◽  
Richard J. Whittington ◽  
...  
Keyword(s):  
Growth Rate ◽  
Quantitative Pcr ◽  
Mycobacterium Avium ◽  
Large Scale ◽  
Liquid Culture ◽  
Vaccine Development ◽  
Culture Method ◽  
Content Type ◽  
Antimicrobial Screening

ABSTRACTDetermining the viability of bacteria is a key outcome ofin vitrocellular infection assays. Currently, this is done by culture, which is problematic for fastidious slow-growing bacteria such asMycobacterium aviumsubsp.paratuberculosis, where it can take up to 4 months to confirm growth. This study aimed to identify an assay that can rapidly quantify the number of viableM. aviumsubsp.paratuberculosiscells in a cellular sample. Three commercially available bacterial viability assays along with a modified liquid culture method coupled with high-throughput quantitative PCR growth detection were assessed. Criteria for assessment included the ability of each assay to differentiate live and deadM. aviumsubsp.paratuberculosisorganisms and their accuracy at low bacterial concentrations. Using the culture-based method,M. aviumsubsp.paratuberculosisgrowth was reliably detected and quantified within 2 weeks. There was a strong linear association between the 2-week growth rate and the initial inoculum concentration. The number of viableM. aviumsubsp.paratuberculosiscells in an unknown sample was quantified based on the growth rate, by using growth standards. In contrast, none of the commercially available viability assays were suitable for use with samples fromin vitrocellular infection assays.IMPORTANCERapid quantification of the viability ofMycobacterium aviumsubsp.paratuberculosisin samples fromin vitrocellular infection assays is important, as it allows these assays to be carried out on a large scale.In vitrocellular infection assays can function as a preliminary screening tool, for vaccine development or antimicrobial screening, and also to extend findings derived from experimental animal trials. Currently, by using culture, it takes up to 4 months to obtain quantifiable results regardingM. aviumsubsp.paratuberculosisviability after anin vitroinfection assay; however, with the quantitative PCR and liquid culture method developed, reliable results can be obtained at 2 weeks. This method will be important for vaccine and antimicrobial screening work, as it will allow a greater number of candidates to be screened in the same amount of time, which will increase the likelihood that a favorable candidate will be found to be subjected to further testing.

scholarly journals Normocyte-binding protein required for human erythrocyte invasion by the zoonotic malaria parasite Plasmodium knowlesi

2016 ◽  
Vol 113 (26) ◽  
pp. 7231-7236 ◽  
Author(s):  
Robert W. Moon ◽  
Hazem Sharaf ◽  
Claire H. Hastings ◽  
Yung Shwen Ho ◽  
Mridul B. Nair ◽  
...  
Keyword(s):  
Vaccine Development ◽  
Plasmodium Knowlesi ◽  
Binding Protein ◽  
Cynomolgus Macaque ◽  
Genomic Analysis ◽  
Human Infection ◽  
Comparative Genomic ◽  
Gene Encoding ◽  
Human Rbcs

The dominant cause of malaria in Malaysia is now Plasmodium knowlesi, a zoonotic parasite of cynomolgus macaque monkeys found throughout South East Asia. Comparative genomic analysis of parasites adapted to in vitro growth in either cynomolgus or human RBCs identified a genomic deletion that includes the gene encoding normocyte-binding protein Xa (NBPXa) in parasites growing in cynomolgus RBCs but not in human RBCs. Experimental deletion of the NBPXa gene in parasites adapted to growth in human RBCs (which retain the ability to grow in cynomolgus RBCs) restricted them to cynomolgus RBCs, demonstrating that this gene is selectively required for parasite multiplication and growth in human RBCs. NBPXa-null parasites could bind to human RBCs, but invasion of these cells was severely impaired. Therefore, NBPXa is identified as a key mediator of P. knowlesi human infection and may be a target for vaccine development against this emerging pathogen.

scholarly journals A Versatile Liquid Culture Method to Control the <i>in Vitro</i> Development of Shoot and Root Apical Meristems of Bamboo Plants

2020 ◽  
Vol 11 (02) ◽  
pp. 262-275
Author(s):  
Most Tanziman Ara ◽  
Taiji Nomura ◽  
Yasuo Kato ◽  
Shinjiro Ogita
Keyword(s):  
Liquid Culture ◽  
Culture Method ◽  
Apical Meristems

scholarly journals Peptides derived from gp43, the most antigenic protein from Paracoccidioides brasiliensis, form amyloid fibrils in vitro: implications for vaccine development

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Thyago R. Cardim-Pires ◽  
Ricardo Sant’Anna ◽  
Debora Foguel
Keyword(s):  
In Silico ◽  
Amyloid Fibrils ◽  
Vaccine Development ◽  
Paracoccidioides Brasiliensis ◽  
In Silico Analysis ◽  
Antigenic Protein ◽  
Amyloid Aggregates ◽  
Important Health ◽  
Potential Vaccine

AbstractFungal infection is an important health problem in Latin America, and in Brazil in particular. Paracoccidioides (mainly P. brasiliensis and P. lutzii) is responsible for paracoccidioidomycosis, a disease that affects mainly the lungs. The glycoprotein gp43 is involved in fungi adhesion to epithelial cells, which makes this protein an interesting target of study. A specific stretch of 15 amino acids that spans the region 181–195 (named P10) of gp43 is an important epitope of gp43 that is being envisioned as a vaccine candidate. Here we show that synthetic P10 forms typical amyloid aggregates in solution in very short times, a property that could hamper vaccine development. Seeds obtained by fragmentation of P10 fibrils were able to induce the aggregation of P4, but not P23, two other peptides derived from gp43. In silico analysis revealed several regions within the P10 sequence that can form amyloid with steric zipper architecture. Besides, in-silico proteolysis studies with gp43 revealed that aggregation-prone, P10-like peptides could be generated by several proteases, which suggests that P10 could be formed under physiological conditions. Considering our data in the context of a potential vaccine development, we redesigned the sequence of P10, maintaining the antigenic region (HTLAIR), but drastically reducing its aggregation propensity.

scholarly journals Peptides derived from gp43, the most antigenic protein from Paracoccidioides brasiliensis, form amyloid fibrils in vitro: implications for vaccine development

2021 ◽  
Author(s):  
Thyago R. Cardim-Pires ◽  
Ricardo Sant’Anna ◽  
Debora Foguel
Keyword(s):  
In Silico ◽  
Amyloid Fibrils ◽  
Vaccine Development ◽  
Paracoccidioides Brasiliensis ◽  
In Silico Analysis ◽  
Antigenic Protein ◽  
Amyloid Aggregates ◽  
Important Health ◽  
Potential Vaccine

Abstract Fungal infection is an important health problem in Latin America, and in Brazil in particular. Paracoccidioides (P. brasiliensis and P. lutzii) is responsible for paracoccidioidomycosis, a disease that affects mainly the lungs. The glycoprotein gp43 is involved in fungi adhesion to epithelial cells, which makes this protein an interesting target of study. A specific stretch of 15 amino acids that spans the region 181-195 (named P10) of gp43 is an important epitope of gp43 that is being envisioned as a vaccine candidate. Here we show that synthetic P10 forms typical amyloid aggregates in solution in very short times, a property that could hamper vaccine development. Seeds obtained by fragmentation of P10 fibrils were able to induce the aggregation of P4, but not P23, two other peptides derived from gp43. In silico analysis revealed several regions within the P10 sequence that are capable of forming amyloid with steric zipper architecture. Besides, in-silico proteolysis studies with gp43 revealed that aggregation-prone, P10-like peptides could be generated by several proteases, which suggests that P10 could be formed under physiological conditions. Considering our data in the context of a potential vaccine development, we redesigned the sequence of P10, maintaining the antigenic region (HTLAIR), but drastically reducing its aggregation propensity.

TWO MACHINES FOR IN VITRO PROPAGATION OF PLANTS IN LIQUID MEDIA

1983 ◽  
Vol 63 (1) ◽  
pp. 311-316 ◽  
Author(s):  
ROBERT E. HARRIS ◽  
EDWIN B. B. MASON
Keyword(s):  
Nicotiana Tabacum ◽  
In Vitro Propagation ◽  
Liquid Culture ◽  
Culture Method ◽  
The Other ◽  
Liquid Media ◽  
Wide Mouth ◽  
Agar Media ◽  
Two Machines

Two machines are described, one to tilt 400 50-mL or 320 125-mL Erlenmeyer flasks, and the other to rock 120 455-mL or 70 910-mL square wide-mouth Mason jars, or combinations of the different container sizes. In grapes, explants transferred to liquid media on the machines after 28 days on agar produced seven times as many shoots in 90 days as explants maintained on agar media. Similar increases were obtained with Arctostaphylos uva ursi, Fuchsia hybrida ’Swingtime,’ Amelanchier alnifolia, and Nicotiana tabacum ’Xanthi-nc.’ The liquid culture method also reduces cost by using less media and agar.Key words: Machines, in vitro, propagation, liquid-media

In Silico Study of Potential Cross-Kingdom Plant MicroRNA Based Regulation in Chronic Myeloid Leukemia

2020 ◽  
Vol 17 (2) ◽  
pp. 125-132
Author(s):  
Marjanu Hikmah Elias ◽  
Noraziah Nordin ◽  
Nazefah Abdul Hamid
Keyword(s):  
Targeted Therapy ◽  
In Silico ◽  
Structure Prediction ◽  
Tertiary Structure ◽  
Target Prediction ◽  
In Silico Analysis ◽  
Microrna Target ◽  
Good Target

Background: Chronic Myeloid Leukaemia (CML) is associated with the BCRABL1 gene, which plays a central role in the pathogenesis of CML. Thus, it is crucial to suppress the expression of BCR-ABL1 in the treatment of CML. MicroRNA is known to be a gene expression regulator and is thus a good candidate for molecularly targeted therapy for CML. Objective: This study aims to identify the microRNAs from edible plants targeting the 3’ Untranslated Region (3’UTR) of BCR-ABL1. Methods: In this in silico analysis, the sequence of 3’UTR of BCR-ABL1 was obtained from Ensembl Genome Browser. PsRNATarget Analysis Server and MicroRNA Target Prediction (miRTar) Server were used to identify miRNAs that have binding conformity with 3’UTR of BCR-ABL1. The MiRBase database was used to validate the species of plants expressing the miRNAs. The RNAfold web server and RNA COMPOSER were used for secondary and tertiary structure prediction, respectively. Results: In silico analyses revealed that cpa-miR8154, csi-miR3952, gma-miR4414-5p, mdm-miR482c, osa-miR1858a and osa-miR1858b show binding conformity with strong molecular interaction towards 3’UTR region of BCR-ABL1. However, only cpa-miR- 8154, osa-miR-1858a and osa-miR-1858b showed good target site accessibility. Conclusion: It is predicted that these microRNAs post-transcriptionally inhibit the BCRABL1 gene and thus could be a potential molecular targeted therapy for CML. However, further studies involving in vitro, in vivo and functional analyses need to be carried out to determine the ability of these miRNAs to form the basis for targeted therapy for CML.

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