| 1. |
- Labiran, Clare, et al.
(författare)
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Genotyping markers used for multi locus VNTR analysis with ompA (MLVA-ompA) and multi sequence typing (MST) retain stability in Chlamydia trachomatis
- 2012
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Ingår i: Frontiers in Cellular and Infection Microbiology. - : Frontiers Media SA. - 2235-2988. ; 2
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Tidskriftsartikel (refereegranskat)abstract
- We aimed to evaluate the stability of the Chlamydia trachomatis multi locus VNTR analysis (MLVA-ompA) and multi sequence typing (MST) systems through multiple passages in tissue culture. Firstly, we analyzed the stability of these markers through adaptation of C. trachomatis to tissue culture and secondly, we examined the stability of a four-locus MLVA-ompA and a five-locus MST system after multiple passages in tissue culture. Marker sequences were monitored through successive chlamydial developmental cycles to evaluate the stability of the individual DNA markers through many bacterial divisions and this, in turn, informed us of the usefulness of using such typing systems for short and long-term molecular epidemiology. Southampton genitourinary medicine (GUM) clinic isolates from endocervical swabs collected from C. trachomatis positive women were passaged through tissue culture. MLVA-ompA typing was applied to primary swab samples and to the same samples after C. trachomatis had been passaged through cell culture (eight passages). Sequence data from time-zero and passage-eight isolates were aligned with reference sequences to determine the stability of the markers. The Swedish new variant (nvCT) underwent 72 passages in cell culture and the markers of the two schemes were similarly analyzed. Analysis of genetic markers of the MLVA-ompA typing system before and after the isolates were introduced to tissue culture showed no change in the dominant sequence. The nvCT that had been passaged 72 times over the duration of a year also showed no variation in the dominant sequence for both the genotyping schemes. MLVA-ompA and MST markers are stable upon adaptation of C. trachomatis to tissue culture following isolation of strains from primary endocervical swab samples. These markers remain stable throughout multiple rounds of cell-division in tissue culture, concomitant with the incubation period and appearance of symptoms normally associated with host-infection. Both genotyping schemes are, therefore, suitable for epidemiology of C. trachomatis.
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| 2. |
- Lai, En Yin, et al.
(författare)
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Pressure induces intracellular calcium changes in juxtaglomerular cells in perfused afferent arterioles
- 2011
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Ingår i: Hypertension Research. - : Springer Science and Business Media LLC. - 0916-9636 .- 1348-4214. ; 34:8, s. 942-948
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Tidskriftsartikel (refereegranskat)abstract
- Calcium (Ca(2+)) has an important role in nearly all types of cellular secretion, with a particularly novel role in the juxtaglomerular (JG) cells in the kidney. In JG cells, Ca(2+) inhibits renin secretion, which is a major regulator of blood pressure and renal hemodynamics. However, whether alterations in afferent arteriolar (Af-Art) pressure change intracellular Ca(2+) concentration ([Ca(2+)](i)) in JG cells and whether [Ca(2+)](i) comes from extracellular or intracellular sources remains unknown. We hypothesize that increases in perfusion pressure in the Af-Art result in elevations in [Ca(2+)](i) in JG cells. We isolated and perfused Af-Art of C57BL6 mice and measured changes in [Ca(2+)](i) in JG cells in response to perfusion pressure changes. The JG cells' [Ca(2+)](i) was 93.3 +/- 2.2 nM at 60 mm Hg perfusion pressure and increased to 111.3 +/- 13.4, 119.6 +/- 7.3, 130.3 +/- 2.9 and 140.8 +/- 12.1 nM at 80, 100, 120 and 140 mm Hg, respectively. At 120 mm Hg, increases in [Ca(2+)](i) were reduced in mice receiving the following treatments: (1) the mechanosensitive cation channel blocker, gadolinium (94.6 +/- 7.5 nM); (2) L-type calcium channel blocker, nifedipine (105.8 +/- 7.5 nM); and (3) calcium-free solution plus ethylene glycol tetraacetic acid (96.0 +/- 5.8 nM). Meanwhile, the phospholipase C inhibitor, inositol triphosphate receptor inhibitor, T-type calcium channel blocker, N-type calcium channel blocker and Ca(2+)-ATPase inhibitor did not influence changes in [Ca(2+)](i) in JG cells. In summary, JG cell [Ca(2+)](i) rise as perfusion pressure increases; furthermore, the calcium comes from extracellular sources, specifically mechanosensitive cation channels and L-type calcium channels.
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| 3. |
- Sigar, Ira M., et al.
(författare)
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Plasmid deficiency in urogenital isolates of Chlamydia trachomatis reduces infectivity and virulence in a mouse model
- 2014
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Ingår i: Pathogens and Disease. - : Wiley-Blackwell. - 2049-632X. ; 70:1, s. 61-69
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Tidskriftsartikel (refereegranskat)abstract
- We hypothesized that the plasmid of urogenital isolates of Chlamydia trachomatis would modulate infectivity and virulence in a mouse model. To test this hypothesis, we infected female mice in the respiratory or urogenital tract with graded doses of a human urogenital isolate of C.trachomatis, serovar F, possessing the cognate plasmid. For comparison, we inoculated mice with a plasmid-free serovar F isolate. Following urogenital inoculation, the plasmid-free isolate displayed significantly reduced infectivity compared with the wild-type strain with the latter yielding a 17-fold lower infectious dose to yield 50% infection. When inoculated via the respiratory tract, the plasmid-free isolate exhibited reduced infectivity and virulence (as measured by weight change) when compared to the wild-type isolate. Further, differences in infectivity, but not in virulence were observed in a C.trachomatis, serovar E isolate with a deletion within the plasmid coding sequence 1 when compared to a serovar E isolate with no mutations in the plasmid. We conclude that plasmid loss reduces virulence and infectivity in this mouse model. These findings further support a role for the chlamydial plasmid in infectivity and virulence in vivo.
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| 4. |
- Wang, Yibing, et al.
(författare)
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Genetic Transformation of a Clinical (Genital Tract), Plasmid-Free Isolate of Chlamydia trachomatis: Engineering the Plasmid as a Cloning Vector
- 2013
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 8:3
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Tidskriftsartikel (refereegranskat)abstract
- Our study had three objectives: to extend the plasmid-based transformation protocol to a clinical isolate of C. trachomatis belonging to the trachoma biovar, to provide "proof of principle'' that it is possible to "knock out'' selected plasmid genes (retaining a replication competent plasmid) and to investigate the plasticity of the plasmid. A recently developed, plasmid-based transformation protocol for LGV isolates of C. trachomatis was modified and a plasmid-free, genital tract C. trachomatis isolate from Sweden (SWFP-) was genetically transformed. Transformation of this non-LGV C. trachomatis host required a centrifugation step, but the absence of the natural plasmid removed the need for plaque purification of transformants. Transformants expressed GFP, were penicillin resistant and iodine stain positive for accumulated glycogen. The transforming plasmid did not recombine with the host chromosome. A derivative of pGFP::SW2 carrying a deletion of the plasmid CDS5 gene was engineered. CDS5 encodes pgp3, a protein secreted from the inclusion into the cell cytoplasm. This plasmid (pCDS5KO) was used to transform C. trachomatis SWFP-, and established that pgp3 is dispensable for plasmid function. The work shows it is possible to selectively delete segments of the chlamydial plasmid, and this is the first step towards a detailed molecular dissection of the role of the plasmid. The 3.6 kb beta-galactosidase cassette was inserted into the deletion site of CDS5 to produce plasmid placZ-CDS5KO. Transformants were penicillin resistant, expressed GFP and stained for glycogen. In addition, they expressed b-galactosidase showing that the lacZ cassette was functional in C. trachomatis. An assay was developed that allowed the visualisation of individual inclusions by X-gal staining. The ability to express active beta-galactosidase within chlamydial inclusions is an important advance as it allows simple, rapid assays to measure directly chlamydial infectivity without the need for plaquing, fluorescence or antibody staining.
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| 5. |
- Wang, Yibing, et al.
(författare)
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Transformation of a plasmid-free, genital tract isolate of Chlamydia trachomatis with a plasmid vector carrying a deletion in CDS6 revealed that this gene regulates inclusion phenotype
- 2013
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Ingår i: Pathogens and Disease. - 2049-632X. ; 67:2, s. 100-103
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Tidskriftsartikel (refereegranskat)abstract
- The development of a plasmid-based genetic transformation protocol for Chlamydia trachomatis provides the basis for the detailed investigation of the function of the chlamydial plasmid and its individual genes or coding sequences (CDS). In this study we constructed a plasmid vector with CDS6 deleted (pCDS6KO) from the original Escherichia coli/C. trachomatis shuttle vector pGFP::SW2. pCDS6KO was transformed into a clinical isolate of C. trachomatis from Sweden that is plasmid-free (C. trachomatis SWFP-). Penicillin-resistant transformants expressing the green fluorescent protein were selected. These transformants did not stain with iodine, indicating that this property is regulated by CDS6 or its gene product. In addition, mature inclusions of C. trachomatis SWFP-transformed by pCDS6KO displayed an identical morphological phenotype to the untransformed plasmid-free recipient host. In this phenotype the morphology of inclusions was altered with the chlamydiae lining the periphery of the inclusion leaving a 'hole' in the centre. These green fluorescent inclusions appear 'doughnut-shaped' with an empty centre when examined under blue light, giving rise to a characteristic 'black hole' phenotype. Our study demonstrates the power of the new genetic system for investigating chlamydial gene function using gene deletion technology.
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| 6. |
- Xu, Xiaobao, et al.
(författare)
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Efficient p-type dye-sensitized solar cells based on disulfide/thiolate electrolytes
- 2013
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Ingår i: Nanoscale. - : Royal Society of Chemistry (RSC). - 2040-3364 .- 2040-3372. ; 5:17, s. 7963-7969
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Tidskriftsartikel (refereegranskat)abstract
- Herein, an organic redox couple 1-methy-1H-tetrazole-5-thiolate (T-) and its disulfide dimer (T-2) redox shuttle, as an electrolyte, is introduced in a p-type dye-sensitized solar cell (DSC) on the basis of an organic dye (P1) sensitizer and nanocrystal CuCrO2 electrode. Using this iodide-free transparent redox electrolyte in conjunction with the sensitized heterojunction, we achieve a high open-circuit voltage of over 300 mV. An optimal efficiency of 0.23% is obtained using a CoS counter electrode and an optimized electrolyte composition under AM 1.5 G 100 mW cm(-2) light illumination which, to the best of our knowledge, represents the highest efficiency that has so far been reported for p-type DSCs using organic redox couples.
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