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The new issue is now available.Journal of Graphic Science of Japan

Vol.49 No.2

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Stability of Reference Genes for Messenger RNA Quantification by Real-Time PCR in Mouse Dextran Sodium Sulfate Experimental Colitis

by Nour Eissa, Hayam Hussein, Hongxing Wang, Mohammad F. Rabbi, Charles N. Bernstein, Jean-Eric Ghia

Background

Many animal models have been developed to characterize the complexity of colonic inflammation. In dextran sodium sulfate (DSS) experimental colitis in mice the choice of reference genes is critical for accurate quantification of target genes using quantitative real time PCR (RT-qPCR). No studies have addressed the performance of reference genes in mice DSS-experimental colitis. This study aimed to determine the stability of reference genes expression (RGE) in DSS-experimental murine colitis.

Methods

Colitis was induced in male C57BL/6 mice using DSS5% for 5 days, control group received water. RNA was extracted from inflamed and non-inflamed colon. Using RT-qPCR, comparative analysis of 13 RGE was performed according to predefined criteria and relative colonic TNF-α and IL-1β gene expression was determined by calculating the difference in the threshold cycle.

Results

Colitis significantly altered the stability of mucosal RGE. Commonly used glyceraldehyde-3-phosphate dehydrogenase (Gapdh), β-actin (Actb), or β2-microglobulin (β2m) showed the highest variability within the inflamed and control groups. Conversely, TATA-box-binding protein (Tbp) and eukaryotic translation elongation factor 2 (Eef2) were not affected by inflammation and were the most stable genes. Normalization of colonic TNF-α and IL-1β mRNA levels was dependent on the reference gene used. Depending on the genes used to normalize the data, statistical significance varied from significant when TBP / Eef2 were used to non-significant when Gapdh, Actb or β2m were used.

Conclusions

This study highlights the appropriate choice of RGE to ensure adequate normalization of RT-qPCR data when using this model. Suboptimal RGE may explain controversial results from published studies. We recommend using Tbp and Eef2 instead of Gapdh, Actb or β2m as reference genes.

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Immunometabolic Status during the Peripartum Period Is Enhanced with Supplemental Zn, Mn, and Cu from Amino Acid Complexes and Co from Co Glucoheptonate

by Fernanda Batistel, Johan S. Osorio, Annarita Ferrari, Erminio Trevisi, Michael T. Socha, Juan J. Loor

The peripartum (or transition) period is the most-critical phase in the productive life of lactating dairy cows and optimal supply of trace minerals through more bioavailable forms could minimize the negative effects associated with this phase. Twenty Holstein cows received a common prepartal diet and postpartal diet. Both diets were partially supplemented with an inorganic (INO) mix of Zn, Mn, and Cu to supply 35, 45, and 6 ppm, respectively, of the diet dry matter (DM). Cows were assigned to treatments in a randomized completed block design, receiving an daily oral bolus with INO or organic trace minerals (AAC) Zn, Mn, Cu, and Co to achieve 75, 65, 11, and 1 ppm supplemental, respectively, in the diet DM. Liver tissue and blood samples were collected throughout the experiment. The lower glutamic-oxaloacetic transaminase concentration after 15 days in milk in AAC cows indicate lower hepatic cell damage. The concentration of cholesterol and albumin increased, while IL-6 decreased over time in AAC cows compared with INO indicating a lower degree of inflammation and better liver function. Although the acute-phase protein ceruloplasmin tended to be lower in AAC cows and corresponded with the reduction in the inflammatory status, the tendency for greater serum amyloid A concentration in AAC indicated an inconsistent response on acute-phase proteins. Oxygen radical absorbance capacity increased over time in AAC cows. Furthermore, the concentrations of nitric oxide, nitrite, nitrate, and the ferric reducing ability of plasma decreased with AAC indicating a lower oxidative stress status. The expression of IL10 and ALB in liver tissue was greater overall in AAC cows reinforcing the anti-inflammatory response detected in plasma. The greater overall expression of PCK1 in AAC cows indicated a greater gluconeogenic capacity, and partly explained the greater milk production response over time. Overall, feeding organic trace minerals as complexed with amino acids during the transition period improved liver function and decreased inflammation and oxidative stress.

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Abdominal Pain, the Adolescent and Altered Brain Structure and Function

by Catherine S. Hubbard, Lino Becerra, Nicole Heinz, Allison Ludwick, Tali Rasooly, Rina Wu, Adriana Johnson, Neil L. Schechter, David Borsook, Samuel Nurko

Irritable bowel syndrome (IBS) is a functional gastrointestinal (GI) disorder of unknown etiology. Although relatively common in children, how this condition affects brain structure and function in a pediatric population remains unclear. Here, we investigate brain changes in adolescents with IBS and healthy controls. Imaging was performed with a Siemens 3 Tesla Trio Tim MRI scanner equipped with a 32-channel head coil. A high-resolution T1-weighted anatomical scan was acquired followed by a T2-weighted functional scan. We used a surface-based morphometric approach along with a seed-based resting-state functional connectivity (RS-FC) analysis to determine if groups differed in cortical thickness and whether areas showing structural differences also showed abnormal RS-FC patterns. Patients completed the Abdominal Pain Index and the GI Module of the Pediatric Quality of Life Inventory to assess abdominal pain severity and impact of GI symptoms on health-related quality of life (HRQOL). Disease duration and pain intensity were also assessed. Pediatric IBS patients, relative to controls, showed cortical thickening in the posterior cingulate (PCC), whereas cortical thinning in posterior parietal and prefrontal areas were found, including the dorsolateral prefrontal cortex (DLPFC). In patients, abdominal pain severity was related to cortical thickening in the intra-abdominal area of the primary somatosensory cortex (SI), whereas HRQOL was associated with insular cortical thinning. Disease severity measures correlated with cortical thickness in bilateral DLPFC and orbitofrontal cortex. Patients also showed reduced anti-correlations between PCC and DLPFC compared to controls, a finding that may reflect aberrant connectivity between default mode and cognitive control networks. We are the first to demonstrate concomitant structural and functional brain changes associated with abdominal pain severity, HRQOL related to GI-specific symptoms, and disease-specific measures in adolescents with IBS. It is possible such changes will be responsive to therapeutic intervention and may be useful as potential markers of disease progression or reversal.

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Optimization of Proton CT Detector System and Image Reconstruction Algorithm for On-Line Proton Therapy

by Chae Young Lee, Hankyeol Song, Chan Woo Park, Yong Hyun Chung, Jin Sung Kim, Justin C. Park

The purposes of this study were to optimize a proton computed tomography system (pCT) for proton range verification and to confirm the pCT image reconstruction algorithm based on projection images generated with optimized parameters. For this purpose, we developed a new pCT scanner using the Geometry and Tracking (GEANT) 4.9.6 simulation toolkit. GEANT4 simulations were performed to optimize the geometric parameters representing the detector thickness and the distance between the detectors for pCT. The system consisted of four silicon strip detectors for particle tracking and a calorimeter to measure the residual energies of the individual protons. The optimized pCT system design was then adjusted to ensure that the solution to a CS-based convex optimization problem would converge to yield the desired pCT images after a reasonable number of iterative corrections. In particular, we used a total variation-based formulation that has been useful in exploiting prior knowledge about the minimal variations of proton attenuation characteristics in the human body. Examinations performed using our CS algorithm showed that high-quality pCT images could be reconstructed using sets of 72 projections within 20 iterations and without any streaks or noise, which can be caused by under-sampling and proton starvation. Moreover, the images yielded by this CS algorithm were found to be of higher quality than those obtained using other reconstruction algorithms. The optimized pCT scanner system demonstrated the potential to perform high-quality pCT during on-line image-guided proton therapy, without increasing the imaging dose, by applying our CS based proton CT reconstruction algorithm. Further, we make our optimized detector system and CS-based proton CT reconstruction algorithm potentially useful in on-line proton therapy.

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HIV Testing among Patients with Presumptive Tuberculosis: How Do We Implement in a Routine Programmatic Setting? Results of a Large Operational Research from India

by Ajay MV Kumar, Devesh Gupta, Ashok Kumar, R. S. Gupta, Avinash Kanchar, Raghuram Rao, Suresh Shastri, MD Suryakanth, Chethana Rangaraju, Balaji Naik, Deepak K. Guddemane, Prashant Bhat, Achuthan Sreenivas Nair, Anthony David Harries, Puneet Dewan

Background

In March 2012, World Health Organization recommended that HIV testing should be offered to all patients with presumptive TB (previously called TB suspects). How this is best implemented and monitored in routine health care settings in India was not known. An operational research was conducted in Karnataka State (South India, population 64 million, accounts for 10% of India’s HIV burden), to test processes and learn results and challenges of screening presumptive TB patients for HIV within routine health care settings.

Methods

In this cross-sectional study conducted between January-March 2012, all presumptive TB patients attending public sector sputum microscopy centres state-wide were offered HIV testing by the laboratory technician, and referred to the nearest public sector HIV counselling and testing services, usually within the same facility. The HIV status of the patients was recorded in the routine TB laboratory form and TB laboratory register. The laboratory register was compiled to obtain the number of presumptive TB patients whose HIV status was ascertained, and the number found HIV positive. Aggregate data on reasons for non-testing were compiled at district level.

Results

Overall, 115,308 patients with presumptive TB were examined for sputum smear microscopy at 645 microscopy centres state-wide. Of these, HIV status was ascertained for 62,847(55%) among whom 7,559(12%) were HIV-positive, and of these, 3,034(40%) were newly diagnosed. Reasons for non-testing were reported for 37,700(72%) of the 52,461 patients without HIV testing; non-availability of testing services at site of sputum collection was cited by health staff in 54% of respondents. Only 4% of patients opted out of HIV testing.

Conclusion

Offering HIV testing routinely to presumptive TB patients detected large numbers of previously-undetected instances of HIV infection. Several operational challenges were noted which provide useful lessons for improving uptake of HIV testing in this important group.

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Non-Temperature Induced Effects of Magnetized Iron Oxide Nanoparticles in Alternating Magnetic Field in Cancer Cells

by Sudath Hapuarachchige, Yoshinori Kato, Ethel J. Ngen, Barbara Smith, Michael Delannoy, Dmitri Artemov

This paper reports the damaging effects of magnetic iron-oxide nanoparticles (MNP) on magnetically labeled cancer cells when subjected to oscillating gradients in a strong external magnetic field. Human breast cancer MDA-MB-231 cells were labeled with MNP, placed in the high magnetic field, and subjected to oscillating gradients generated by an imaging gradient system of a 9.4T preclinical MRI system. Changes in cell morphology and a decrease in cell viability were detected in cells treated with oscillating gradients. The cytotoxicity was determined qualitatively and quantitatively by microscopic imaging and cell viability assays. An approximately 26.6% reduction in cell viability was detected in magnetically labeled cells subjected to the combined effect of a static magnetic field and oscillating gradients. No reduction in cell viability was observed in unlabeled cells subjected to gradients, or in MNP-labeled cells in the static magnetic field. As no increase in local temperature was observed, the cell damage was not a result of hyperthermia. Currently, we consider the coherent motion of internalized and aggregated nanoparticles that produce mechanical moments as a potential mechanism of cell destruction. The formation and dynamics of the intracellular aggregates of nanoparticles were visualized by optical and transmission electron microscopy (TEM). The images revealed a rapid formation of elongated MNP aggregates in the cells, which were aligned with the external magnetic field. This strategy provides a new way to eradicate a specific population of MNP-labeled cells, potentially with magnetic resonance imaging guidance using standard MRI equipment, with minimal side effects for the host.

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