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1.
FEBS Lett ; 593(23): 3304-3315, 2019 12.
Article in English | MEDLINE | ID: mdl-31432499

ABSTRACT

The haematopoietic system is established during embryonic life through a series of developmental steps that culminates with the generation of haematopoietic stem cells. Characterisation of the transcriptional network that regulates blood cell emergence has led to the identification of transcription factors essential for this process. Among the many factors wired within this complex regulatory network, ETV2, SCL and RUNX1 are the central components. All three factors are absolutely required for blood cell generation, each one controlling a precise step of specification from the mesoderm germ layer to fully functional blood progenitors. Insight into the transcriptional control of blood cell emergence has been used for devising protocols to generate blood cells de novo, either through reprogramming of somatic cells or through forward programming of pluripotent stem cells. Interestingly, the physiological process of blood cell generation and its laboratory-engineered counterpart have very little in common.


Subject(s)
Blood Cells/metabolism , Core Binding Factor Alpha 2 Subunit/genetics , T-Cell Acute Lymphocytic Leukemia Protein 1/genetics , Transcription Factors/genetics , Blood Cells/cytology , Cell Differentiation/genetics , Cellular Reprogramming/genetics , Hematopoietic Stem Cells , Humans , Mesoderm/growth & development , Mesoderm/metabolism , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolism , Transcriptional Activation
2.
Vet Microbiol ; 203: 280-285, 2017 May.
Article in English | MEDLINE | ID: mdl-28619158

ABSTRACT

Mycobacterial interspersed repetitive unit and variable number tandem repeat (MIRU-VNTR) has been developed as a simple, rapid and cost efficient molecular typing method to differentiate Mycobacterium avium subspecies paratuberculosis (MAP) isolates. The aim of this study was to determine the genomic diversity of MAP across the Republic of Ireland by utilising the MIRU-VNTR typing method on a large collection of MAP isolates. A total of 114 MAP isolates originated from 53 herds across 19 counties in the Republic of Ireland were genotyped based on eight established MIRU-VNTR loci. Four INMV groups were observed during this study. INMV 1 was found in 67 MAP isolates (58.8%) and INMV 2 was observed in 45 isolates (39.4%). INMV 3 and INMV 116 recorded only one isolate each (0.9%). The unique INMV 116 group has never been reported among herds thus far and the molecular pattern of the MAP isolate classified in INMV 116 showed a difference at the MIRU-VNTR X3 locus compared to the other three INMV groups observed. INMV 1, INMV 2 and INMV 3 are observed frequently in Europe and comprised 99.1% of the total MAP isolates characterised in this study, indicating that MAP exhibited low level of genetic diversity across the Republic of Ireland using the MIRU-VNTR method. By the implementation of SNP analysis or MLSSR as an additional typing method, MAP genetic diversity would increase. INMV 3 is unique to Ireland and whereas INMV 116 has never been previously reported among herds by MIRU-VNTR typing.


Subject(s)
Cattle Diseases/microbiology , Genetic Variation , Mycobacterium avium subsp. paratuberculosis/genetics , Paratuberculosis/microbiology , Animals , Cattle , Cattle Diseases/epidemiology , Genotype , Ireland/epidemiology , Minisatellite Repeats/genetics , Mycobacterium avium subsp. paratuberculosis/isolation & purification , Paratuberculosis/epidemiology
3.
Vet Microbiol ; 204: 104-109, 2017 May.
Article in English | MEDLINE | ID: mdl-28532788

ABSTRACT

The aim of this study was to investigate the efficacy of IS_MAP04 as a potential new diagnostic quantitative PCR (qPCR) target for the detection of Mycobacterium avium subspecies paratuberculosis from bovine faeces. IS_MAP04 primers were designed and tested negative against non-MAP strains. The detection limit of IS_MAP04 qPCR was evaluated on different MAP K-10 DNA concentrations and on faecal samples spiked with different MAP K-10 cell dilutions. A collection of 106 faecal samples was analysed and the efficacy of IS_MAP04 was statistically compared with IS900 and IS_MAP02. The detection limits observed for IS_MAP04 and IS900 on MAP DNA was 34 fg and 3.4 fg respectively. The detection limit of MAP from inoculated faecal samples was 102 CFU/g for both IS_MAP04 and IS900 targets and a detection limit of 102 CFU/g was also achieved with a TaqMan qPCR targeting IS_MAP04. The efficacy of IS_MAP04 to detect positive MAP faecal samples was 83.0% compared to 85.8% and 83.9% for IS900 and IS_MAP02 respectively. Strong kappa agreements were observed between IS_MAP04 and IS900 (κ=0.892) and between IS_MAP04 and IS_MAP02 (κ=0.897). As a new molecular target, IS_MAP04 showed that the detection limit was comparable to IS900 to detect MAP from inoculated faecal material. The MAP detection efficacy of IS_MAP04 from naturally infected faecal samples proved to be relatively comparable to IS_MAP02, but yielded efficacy results slightly less than IS900. Moreover, IS_MAP04 could be of significant value when used in duplex or multiplex qPCR assays.


Subject(s)
Bacteriological Techniques/veterinary , Feces/microbiology , Mycobacterium avium subsp. paratuberculosis/genetics , Polymerase Chain Reaction/methods , Animals , Bacteriological Techniques/methods , Cattle , DNA, Bacterial/genetics , Genome, Bacterial , Limit of Detection , Mycobacterium avium subsp. paratuberculosis/isolation & purification , Sensitivity and Specificity
4.
J Microbiol Methods ; 136: 40-48, 2017 05.
Article in English | MEDLINE | ID: mdl-28285167

ABSTRACT

The inability of Mycobacterium avium subspecies paratuberculosis (MAP) to produce endogenous mycobactin in-vitro is most likely due to the presence of a truncated mbtA gene within the mycobactin cluster of MAP. The main goal of this study was to investigate this unique mbtA truncation as a potential novel PCR diagnostic marker for MAP. Novel primers were designed that were located within the truncated region and the contiguous MAP2179 gene. Primers were evaluated against non-MAP isolates and no amplicons were generated. The detection limit of this mbtA-MAP2179 target was evaluated using a range of MAP DNA concentrations, MAP inoculated faecal material and 20 MAP isolates. The performance of mbtA-MAP2179 was compared to the established f57 target. The detection limits recorded for MAP K-10 DNA and from MAP K-10 inoculated faecal samples were 0.34pg and 104CFU/g respectively for both f57 and mbtA-MAP2179. A detection limit of 103CFU/g was recorded for both targets, but not achieved consistently. The detection limit of MAP from inoculated faecal material was successful at 103CFU/g for mbtA-MAP2179 when FAM probe real-time PCR was used. A MAP cell concentration of 102CFU/g was detected successfully, but again not consistently achieved. All 20 mycobacterial isolates were successfully identified as MAP by f57 and mbtA-MAP2179. Interestingly, the mbtA-MAP2179 real-time PCR assay resulted in the formation of a unique melting curve profile that contained two melting curve peaks rather than one single peak. This melting curve phenomenon was attributed towards the asymmetrical GC% distribution within the mbtA-MAP2179 amplicon. This study investigated the implementation of the mbtA-MAP2179 target as a novel diagnostic marker and the detection limits obtained with mbtA-MAP2179 were comparable to the established f57 target, making the mbtA-MAP2179 an adequate confirmatory target. Moreover, the mbtA-MAP2179 target could be implemented in multiplex real-time PCR assays and with its unique melting curve profile adds increased specificity to MAP diagnostic tests.


Subject(s)
Bacterial Proteins/genetics , Biomarkers/analysis , Ligases/genetics , Mycobacterium avium subsp. paratuberculosis/genetics , Mycobacterium avium subsp. paratuberculosis/isolation & purification , Oxazoles/chemistry , Real-Time Polymerase Chain Reaction/methods , Animals , Base Composition , Cattle , Cattle Diseases/diagnosis , Cattle Diseases/microbiology , DNA Primers/genetics , DNA, Bacterial/genetics , Escherichia coli/genetics , Feces/microbiology , Limit of Detection , Mycobacterium/enzymology , Mycobacterium/genetics , Mycobacterium/isolation & purification , Mycobacterium/pathogenicity , Mycobacterium avium subsp. paratuberculosis/enzymology , Mycobacterium avium subsp. paratuberculosis/pathogenicity , Paratuberculosis/diagnosis , Real-Time Polymerase Chain Reaction/veterinary , Sensitivity and Specificity
5.
Endoscopy ; 48(5): 477-483, 2016 May.
Article in English | MEDLINE | ID: mdl-27042930

ABSTRACT

BACKGROUND AND STUDY AIMS: Targeted delivery of specific chemotherapeutic drugs into tumors can be achieved by delivering electrical pulses directly to the tumor tissue. This causes a transient formation of pores in the cell membrane that enables passive diffusion of normally impermeant drugs. A novel device has been developed to enable the endoscopic delivery of this tumor permeabilizing treatment. The aim of the preclinical studies described here was to investigate the efficacy and safety of this nonthermal ablation system in the treatment of gastrointestinal cancer models. METHODS: Murine, porcine, and canine gastrointestinal tumors and tissues were used to assess the efficacy and safety of electroporation delivered through the special device in combination with bleomycin. Tumor cell death, volume, and overall survival were recorded. RESULTS: Murine tumors treated with electrochemotherapy showed excellent responses, with cell death being induced rapidly, mainly via an apoptotic-type mechanism. Use of the system in canine gastrointestinal cancers demonstrated successful local endoluminal tumor resolution, with no safety or adverse effects noted. CONCLUSIONS: Electroporation via the new device in combination with bleomycin offers a nonthermal tumor ablative approach, and presents clinicians with a new option for the management of gastrointestinal cancers.


Subject(s)
Bleomycin/administration & dosage , Drug Delivery Systems , Electrochemotherapy/methods , Electroporation , Endoscopy, Gastrointestinal/methods , Gastrointestinal Neoplasms/drug therapy , Animals , Antibiotics, Antineoplastic/administration & dosage , Cell Line, Tumor , Disease Models, Animal , Dogs , Drug Delivery Systems/instrumentation , Drug Delivery Systems/methods , Electroporation/instrumentation , Electroporation/methods , Mice , Swine , Treatment Outcome
6.
Am J Trop Med Hyg ; 76(4): 626-30, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17426160

ABSTRACT

Sexual stages of Plasmodium falciparum play a key role in the transmission of malaria. Studies on gametocytes are generally based on microscopic detection, but more sensitive detection methods for P. falciparum gametocytes frequently detect sub-patent gametocytes. We used Pfs25 mRNA quantitative-nucleic acid sequence-based amplification (QT-NASBA) to quantify gametocytes in 412 samples from a cross-sectional study in Burkina Faso, covering all age groups, to determine age-related patterns in gametocyte carriage and gametocyte density. The more sensitive QT-NASBA technique gave estimates of gametocyte prevalence 3.3-fold higher than microscopy (70.1% versus 21.4%, respectively). Prevalence of gametocytes significantly decreased with age. Our data suggest that asexual parasite densities are primarily responsible for the age-related decrease of gametocyte prevalence, possibly because of developing asexual stage immunity. Gametocyte densities decrease also with age, primarily because of decreasing asexual parasite densities; only a small but significant age effect on gametocyte density may be caused by developing sexual stage-specific immunity.


Subject(s)
Malaria, Falciparum/epidemiology , Malaria, Falciparum/parasitology , Plasmodium falciparum/growth & development , Plasmodium falciparum/isolation & purification , Protozoan Proteins/isolation & purification , Adolescent , Adult , Age Distribution , Animals , Burkina Faso/epidemiology , Child , Child, Preschool , Cross-Sectional Studies , Humans , Self-Sustained Sequence Replication
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