The Benefits of Using Multi Gauge Software Fujifilm Version 20 for Gel Image Analysis
Figure 1 shows the DNA binding ability of the purified MsDps2 upon incubation with a plasmid DNA (pGEM plasmid 2.9 kb). It can be seen from the gel (lane 2) that the protein binds to DNA. This mode of binding for Dps proteins has been studied earlier, wherein the protein, upon DNA addition forms a huge protein-DNA complex that gets retained in the wells of an agarose gel , , , . As the size of the complex is very big, we did not make any attempt to resolve the complex by other methods. Upon quantification of the band intensities using multigauge software, the amount of DNA was found to be more in lane 2, as compared to the free protein alone in lane 3 and values have been mentioned in the figure legend of figure 1. We inferred that even in lane 3 where no external DNA was added MsDps2 had DNA associated with it. Expectedly, as no added DNA was present, the intensity of DNA in lane 3 is less as compared to that in lane 2. Lane 1 containing the free DNA was used as control and therefore no DNA is present in the well. Further comparison of the DNA binding activity of the full length and deleted protein has been performed through AFM analysis and transmission electron microscopic studies as discussed below.
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In order to isolate and characterize a transcription complex at msdps2 promoter, we need to identify the upstream promoter sequence first. However, it was difficult to characterize the same with in vivo primer extension method , as we do not know under what condition the transcription of msdps2 is activated. Thus, we cloned a 778 base pair upstream sequence of DNA from the translational initiation site of the msdps2 gene, in mc2155 genomic DNA sequence, assuming that the promoter element will be a part of the same. Preliminary multiple round in vitro transcription on this template showed appreciable RNA product with the core RNA polymerase of M. smegmatis reconstituted with M. tuberculosis σA and σB factors (not shown). We have shown before that both M. tuberculosis σA and σB share significant sequence homology with that of M. smegmatis . The DNA fragment was then immobilized on streptavidin coated agarose beads (Sigma Aldrich). We reconstituted M. smegmatis core RNA polymerase with different concentration of σA or σB, named as EσA and EσB respectively and carried out pull-down experiments and probed with antibodies against the respective sigma factors. The western blots were scanned using Multi Gauge V2.3 software (in silico) and the transcript band intensities were quantitated through densitometry. In order to quantitate the differential affinity of the two sigma factors reconstituted holo RNA polymerases namely EσA and EσB for msdps2 promoter, a calibration curve was constructed (fig. 6). Table 1 shows the comparative affinity for EσA and EσB towards msdps2 promoter. One should note that we have not analyzed the association of any ECF sigma factor reconstituted RNA polymerases with msdps2 promoter. However, the above experiment has been done to compare the sigma factor specificity of msdps2 promoter vis-à-vis msdps1 promoter, where msdps1 promoter is transcribed by RNA polymerase containing ECF sigma factors only . The presence of any other sigma factors association with msdps2 promoter, apart from the principle sigma factors, need to be explored.
Semi-quantitative analysis of 3H-PiB, 3H-THK5117 autoradiography and Amylo-Glo and AT8 staining. Box plot with a line at the median represent: a: Specific binding of 3H-PIB and for AD, DS and CN in HIPP and MFG. b: Amylo-Glo density for AD, DS and CN in HIPP and MFG. c: Specific binding of 3H-THK5117 for AD, DS and CN in HIPP and MFG. d: AT8 density for AD, DS and CN in HIPP and MFG. Specific binding for 3H-PIB and 3H-THK5117 is represented in fmol/mg. Gray matter was delineated manually using multigauge software. Density values for Amylo-Glo and AT8 are presented in b and d respectively. The red dots in each plot represent two DS-MCI cases. The empty blue circles in AD represent the EOAD
: Semi-quantitative analyses of 3H-PIB, 3H-THK5117 autoradiography. Box and whiskers plot showing all data point represent: a: Specific binding of 3H-THK5117 in EOAD, LOAD, DS-AD, CN AD, in HIPP and MFG. b: Specific binding of 3H-PIB in EOAD, LOAD, DS-AD, CN AD, in HIPP and MFG. Specific binding for 3H-PIB and 3H-THK5117 is represented in fmol/mg. Gray matter was delineated manually using multigauge software.
Primers located directly adjacent to the exon 10 c.1020*+718G>A SNP (forward primer, TTAAGTCCAGCTTGGCCAAG; and reverse primer, TGTAGAGATGGGATTTCACCA) were then used in single-cycle PCR reactions incorporating radiolabelled dNTPs. Both forward and reverse reactions were performed to verify results. Radiolabelled fragments were separated on a 10% denaturing acrylamide gel, and were visualised by autoradiography . Densitometric analysis was performed using Fujifilm Multigauge 2003 software (Fuji Photo Film Co., Tokyo, Japan).
You might have noticed that I wrote "works well for the most part" in the previous paragraph. I did run into one exception: an inexpensive Canon multifunction printer. None of the software I used could detect it. I had to download and install Canon's Linux scanner software, which did work.
Recently, these pioneering studies paved the way for a commercially available rodent 3DE system that allows automated respiratory-gated acquisition of high-resolution 2D B-Mode images at different levels of the heart and at every point of the cardiac cycle (Fig. 1a-c) [16, 17]. 3DE data sets are built by tomographic multi-slice reconstruction of acquired 2D images up to a step size of 50 μm, and can be analyzed with a dedicated software package allowing visualization and calculation of LV volumes along the cardiac cycle (Fig. 1d-f).
Phantoms were scanned in a 3 Tesla small animal magnetic resonance system (MR Solutions, Guildford, United Kingdom) with a quadrature birdcage cardiac volume coil as previously reported by us . A T2-weighed fast spin echo sequence with following parameters was applied: repetition time, 4800 ms; echo time, 68 ms; flip angle, 90; field of view, 40.00\40.00\ 0.30 mm; pixel spacing 0.16\0.16; number of signal averages, 3; slice thickness 0.3 mm. Volumes were calculated by multi-slice tracing using Osirix software (version 7.0.3; Pixmeo SARL, Geneva, Switzerland).
Our results demonstrate that 3DE is suitable to determine cardiac volumes in vivo. These findings are in line with pioneering studies, evaluating non-commercially available 3DE-techniques [14, 15]. In 1999, Scherrer-Crosbie and colleagues demonstrated for the first time that multidimensional imaging allows precise LV volumetry and ventricular function, comparable to flow-probe measurements in a mouse model of myocardial infarction . Dawson et al. applied ECG- and respiration-gated 3DE in small animals and were the first, who demonstrated excellent agreement by comparison with the current gold standard volumetric technology (MRI) . However, the widespread application of these non-commercially 3DE approaches was limited with regards to standardization, post-processing software and spatial resolutions. Based on these pioneering studies, the present commercially available 3DE system for small animals was launched . Very recently, Damen and colleagues analyzed the novel commercially available 3DE-system in a genetic model of LV hypertrophy and healthy controls in comparison to 1DE and CMR . The authors found no significant differences between 3DE and CMR measured mean values of cardiac volumetry and corresponding relative metrics, whereas 1DE on average overestimated cardiac volumes . In contrast, our results demonstrated a moderate overestimation of 3DE-assessed cardiac volumes when compared to CMR values. This effect might be explained by the differences in step size used for 3DE (step size: 0.1 mm) and CMR (step size: 1.0 mm) analysis in our study. A reduction of CMR-slice thickness will increase spatiotemporal resolution of the acquired images, but will consequently lead to prolonged acquisition time, which further can cause problems with anesthesia. It is known from the clinics that a coarsely chosen resolution of CMR-image lines can lead to partial volume effects, in case the last part of the apex (short axis orientation) is located between two slices and therefore not included during endocardial border tracing [28, 29]. This effect has already been reported for other imaging techniques like positron emission tomography (PET) in preclinical animal models [30, 31]. In terms of our findings, the 10-fold difference in resolution between 3DE and CMR may lead to an ostensible overestimation of 3DE-assessed volumes, but might also be reasoned by a CMR-based partial volume effect. Our findings are in contrast to the data of Damen and colleagues, who detected no significant differences for mean values of cardiac volumes , although they also used different slice thicknesses during image acquisition (3DE: 0.076 mm vs CMR: 1.00 mm).
First, all echocardiographic examinations were performed under inhaled anesthesia which might have had an impact on heart rate and function and hampers comparison to CMR-assessed values. Further, echocardiographic examination, including novel 3DE, is always limited due to sternum, rib and lung artifacts, which can blur endocardial borders. Second, the choice of end-diastolic, mid-systolic and end-systolic time periods during the cardiac cycle is automatically done by the VevoLab software. Therefore, the user is dependent on the correct selection with no option for the user to validate the choice of cardiac cycle time periods. This may become relevant when investigating cardiac pathologies with arrhythmias. Third, the sample size of the present study was relatively low and only two animal cohorts were used to evaluate novel 3DE. Thus, future validation using larger sample sizes and different animal models is still required. Fourth, tracing of MRI/CMR data was performed manually, whereas ultrasound images were analyzed with semiautomatic software tools. Fifth, we found moderate inter- and intra-observer variabilities for 3DE in diseased mice, which were comparable or slightly inferior when compared to conventional echocardiography. Sixth, we only acquired images from mice during a single ultrasound session. Future studies will reveal reproducibility of novel 3DE when screening the same animal in multiple ultrasound sessions. Lastly, it should be taken into account that we used a suboptimal setting of body temperature controlling during image acquisition and did not monitor body temperature of mice directly. Therefore, we cannot prove whether body temperature variations had potential confounding effects on the assessment of cardiac volumetry in our study.