Abstract 12423: Normoxic Therapy Attenuated Oxidative Stress Related mRNA Gene Expressions in a Post-Cardiac Arrest Rat Model

Objective: We recently reported that post-resuscitation normoxic therapy attenuates oxidative stress in multiple organs and improves post-cardiac arrest (CA) organ injury, oxygen metabolism, and survival. Yet, detailed mechanisms of gene expression patterns and signaling pathways mitigated by normoxic therapy have not been elucidated. Therefore, we assessed post-resuscitation normoxic therapy-modified gene expression of oxidative stress-related signaling molecules. Methods: Rats were resuscitated from 10 minutes of asphyxial CA and divided into 2 groups: those that inhaled 100% supplemental O 2 (CA-FIO2 1.0) and those that inhaled 30% supplemental O 2 (CA-FIO2 0.3). Control groups were also prepared for comparison (control-FIO2 1.0, control-FIO2 0.3). At 2 hours after resuscitation, brain and heart tissues were collected, and mRNA purifications followed by real-time PCR measurements were performed to compare gene expression of hyperoxia-induced inflammatory and apoptosis-related signaling pathways amongst these groups. Results: In the brain, relative IL-1 beta mRNA gene expression levels, which represent inflammatory signaling pathways, increased post-CA (8.1±2.3 in CA-FIO2 1.0 and 1.0±0.4 in control-FIO2 0.3, p<0.05), but were significantly attenuated by normoxic therapy (2.3±0.2 in CA-FIO2 0.3, p<0.05). Likewise, normoxic therapy significantly reduced oxidative stress-induced inflammatory (NFKB1, TGFB1, MAPK14, TRAF6) and apoptosis-related (BAX, EGF) mRNA gene expression levels in the brain, whereas no statistical differences were detected in the heart. Conclusions: Post-CA normoxic therapy significantly attenuated the gene expression of oxidative stress-induced inflammation and apoptosis in the brain, while there were no remarkable changes in the heart. Therefore, it is inferred that the heart is more tolerant to hyperoxic injury compared to the brain.

The Impact of CB1 Receptor on Nuclear Receptors in Skeletal Muscle Cells

Cannabinoids are abundant signaling compounds; their influence predominantly arises via engagement with the principal two G-protein-coupled cannabinoid receptors, CB1 and CB2. One suggested theory is that cannabinoids regulate a variety of physiological processes within the cells of skeletal muscle. Earlier publications have indicated that expression of CB1 receptor mRNA and protein has been recognized within myotubes and tissues of skeletal muscle from both murines and humans, thus representing a potentially significant pathway which plays a role in the control of skeletal muscular activities. The part played by CB1 receptor activation or inhibition with respect to these functions and relevant to targets in the periphery, especially skeletal muscle, is not fully delineated. Thus, the aim of the current research was to explore the influence of CB1 receptor stimulation and inhibition on downstream signaling of the nuclear receptor, NR4A, which regulates the immediate impacts of arachidonyl-2’-chloroethylamide (ACEA) and/or rimonabant in the cells of skeletal muscle. Murine L6 skeletal muscle cells were used in order to clarify additional possible molecular signaling pathways which contribute to alterations in the CB1 receptor. Skeletal muscle cells have often been used; it is well-documented that they express cannabinoid receptors. Quantitative real-time probe-based polymerase chain reaction (qRT-PCR) assays are deployed in order to assess the gene expression characteristics of CB1 receptor signaling. In the current work, it is demonstrated that skeletal muscle cells exhibit functional expression of CB1 receptors. This can be deduced from the qRT-PCR assays; triggering CB1 receptors amplifies both NR4A1 and NR4A3 mRNA gene expression. The impact of ACEA is inhibited by the selective CB1 receptor antagonist, rimonabant. The present research demonstrated that 10 nM of ACEA notably amplified mRNA gene expression of NR4A1 and NR4A3; this effect was suppressed by the addition of 100 nM rimonabant. Furthermore, the CB1 receptor antagonist led to the downregulation of mRNA gene expression of NR4A1, NR4A2 and NR4A3. In conclusion, in skeletal muscle, CB1 receptors were recognized to be important moderators of NR4A1 and NR4A3 mRNA gene expression; these actions may have possible clinical benefits. Thus, in skeletal muscle cells, a possible physiological expression of CB1 receptors was identified. It is as yet unknown whether these CB1 receptors contribute to pathways underlying skeletal muscle biological function and disease processes. Further research is required to fully delineate their role(s).

Zinc Status Index (ZSI) for Quantification of Zinc Physiological Status

Zinc (Zn) deficiency is estimated to affect over one billion (17%) of the world’s population. Zn plays a key role in various cellular processes such as differentiation, apoptosis, and proliferation, and is used for vital biochemical and structural processes in the body. Widely used biomarkers of Zn status include plasma, whole blood, and urine Zn, which decrease in severe Zn deficiency; however, accurate assessment of Zn status, especially in mild to moderate deficiency, is difficult, as studies with these biomarkers are often contradictory and inconsistent. Thus, sensitive and specific biological markers of Zn physiological status are still needed. In this communication, we provide the Zn status index (ZSI) concept, which consists of a three-pillar formula: (1) the LA:DGLA ratio, (2) mRNA gene expression of Zn-related proteins, and (3) gut microbiome profiling to provide a clear assessment of Zn physiological status and degree of Zn deficiency with respect to assessing dietary Zn manipulation. Analysis of five selected studies found that with lower dietary Zn intake, erythrocyte LA:DGLA ratio increased, mRNA gene expression of Zn-related proteins in duodenal and liver tissues was altered, and gut microbiota populations differed, where the ZSI, a statistical model trained on data from these studies, was built to give an accurate estimation of Zn physiological status. However, the ZSI needs to be tested and refined further to determine its full potential.

The Correlation Between Tnm and Yy1 and P53 Mrna Expression in Nasopharyngeal Cancer

Nasopharyngeal cancer is the fifth most severe malignant disease in the head and neck in the human body. The main treatment is chemoradio therapy. The protein gene 53 (p53) is a tumor suppressor gene. YinYang1 (YY1) is a transcription factor having an important role in cell cycle control. YY1 can function as an activator, suppressor or initiator of the gene transcription process. This research aims to see the relationship between p53 mRNA gene expression and YY1 mRNA gene expression on the NPC TNM Stage. Materials andmethods cross-sectional research at 20 WHO type 3 NPC in which 3 samples after chemoradiotherapy, 17 non-chemoradio therapy samples consisted of 8 stage two samples, 7 stage three samples and 2 stage four samples. With RT-PCR, YY1 mRNA gene expression and p53 mRNA gene expression were measured, then the T-test was independent of the average chemoradio therapy group. It was concluded, at a higher NPC stage, the level of YY1 mRNA gene expression was relatively higher while p53 expression was lower. Post-chemoradio therapy levels of p53 mRNA gene expression were higher and YY1 expression was lower than in the non-chemoradio therapy group.

Safety and Efficacy of Felid Herpesvirus-1 Deletion Mutants in Cats

Felid herpesvirus-1 (FeHV-1) is an important respiratory and ocular pathogen of cats and current vaccines are limited in duration and efficacy because they do not prevent infection, viral nasal shedding and latency. To address these shortcomings, we have constructed FeHV-1 gE-TK- and FeHV-1 PK- deletion mutants (gE-TK- and PK-) using bacterial artificial chromosome (BAC) mutagenesis and shown safety and immunogenicity in vitro. Here, we compare the safety and efficacy of a prime boost FeHV-1 gE-TK- and FeHV-1 PK- vaccination regimen with commercial vaccination in cats. Cats in the vaccination groups were vaccinated at 3-week intervals and all cats were challenge infected 3 weeks after the last vaccination. Evaluations included clinical signs, nasal shedding, virus neutralizing antibodies (VN), cytokine mRNA gene expression, post-mortem histology and detection of latency establishment. Vaccination with gE-TK- and PK- mutants was safe and resulted in significantly reduced clinical disease scores, pathological changes, viral nasal shedding, and viral DNA in the trigeminal ganglia (the site of latency) following infection. Both mutants induced VN antibodies and interferons after immunization. In addition, after challenge infection, we observed a reduction of IL-1β expression, and modulation of TNFα, TGFβ and IL10 expression. In conclusion, this study shows the merits of using FeHV-1 deletion mutants for prevention of FeHV-1 infection in cats.

L-carnitine suppresses cisplatin-induced renal injury in rats: impact on cytoskeleton proteins expression

We designed this work to examine the curative role of L-carnitine (LCAR) in a rat model of cisplatin (CDDP)-induced kidney injury. We induced kidney injury in rats by a single intraperitoneal injection of 5 mg/kg of CDDP. Fifteen days post injection, rats were orally supplemented with 354 mg/kg of LCAR for another 15 days. Kidney tissues were subjected to histo-biochemical analysis along with mRNA gene expression quantification for cytoskeleton proteins encoding genes (vimentin, nestin, and connexin 43) by real-time reverse transcription polymerase chain reaction. LCAR reversed CDDP-induced renal structural and functional impairments. LCAR significantly declined serum urea and creatinine concentrations, restored oxidant/antioxidant balance, reversed inflammation, and antagonized caspase 3-mediated apoptotic cell death in renal tissues. Moreover, LCAR effectively down-regulated cytoskeleton proteins mRNA levels, reflecting amelioration of CDDP-provoked podocyte injury. We concluded that LCAR has a favorable therapeutic utility against CDDP-induced kidney injury.

Diet-Induced Obesity Disrupts Trace Element Homeostasis and Gene Expression in the Olfactory Bulb

The aim of this study was to determine the impact of diet-induced obesity (DIO) on trace element homeostasis and gene expression in the olfactory bulb and to identify potential interaction effects between diet, sex, and strain. Our study is based on evidence that obesity and olfactory bulb impairments are linked to neurodegenerative processes. Briefly, C57BL/6J (B6J) and DBA/2J (D2J) male and female mice were fed either a low-fat diet or a high-fat diet for 16 weeks. Brain tissue was then evaluated for iron, manganese, copper, and zinc concentrations and mRNA gene expression. There was a statistically significant diet-by-sex interaction for iron and a three-way interaction between diet, sex, and strain for zinc in the olfactory bulb. Obese male B6J mice had a striking 75% increase in iron and a 50% increase in manganese compared with the control. There was an increase in zinc due to DIO in B6J males and D2J females, but a decrease in zinc in B6J females and D2J males. Obese male D2J mice had significantly upregulated mRNA gene expression for divalent metal transporter 1, alpha-synuclein, amyloid precursor protein, dopamine receptor D2, and tyrosine hydroxylase. B6J females with DIO had significantly upregulated brain-derived neurotrophic factor expression. Our results demonstrate that DIO has the potential to disrupt trace element homeostasis and mRNA gene expression in the olfactory bulb, with effects that depend on sex and genetics. We found that DIO led to alterations in iron and manganese predominantly in male B6J mice, and gene expression dysregulation mainly in male D2J mice. These results have important implications for health outcomes related to obesity with possible connections to neurodegenerative disease.

Risk Assessment in Hereditary Colorectal Cancer Family by Using APC and MSH2 mRNA Gene Expression and Bayesian Analysis

BACKGROUND: Some of colorectal cancers (CRCs) are familial, however, heterozygote relatives have approximately 80% lifetime risk of cancer. Risk assessment of CRC’s family could be calculated by direct measurement of mRNA gene expression and Bayesian theorem which is modifying initial background of pedigree risk with additional conditional information. This application has not been reported.METHODS: The cross-sectional translational sequential studies were performed: (1) adenomatous polyposis coli (APC) and MutS homolog (MSH)2 mRNA quantitative RT PCR gene expressions in tissue and whole blood CRC patients; (2) gene expression was determined in matched controls; and (3) pedigree and Bayesian analysis was calculated in the patient’s family of Proband.RESULTS: Fourty CRC and 31 control subjects were enrolled. The mean blood APC level control’s group was 13,261±670 fold-change (fc) and blood MSH2 level was 12,219±756 fc. The cut-off points for hereditary APC was 12,195 fc and MSH2 was 11,059 fc. The mean APC blood level in CRC subject was 11,578±2,638 fc and MSH2 blood level was 11,411±2,912 fc. There were significant differences APC and MSH level between tissue and blood level in CRC. Eight of 40 CRC subjects had a history of familial CRC. Four patients and 10 Probands were available for recurrence risk evaluation of pedigree analysis, RNA PCR quantitative and Bayesian calculation.CONCLUSION: There was determined a cut-off point of hereditary mRNA quantitative expression. The APC and MSH2 levels in CRC subjects were significantly lower than controls. Bayesian analysis allowed for the calculation of relative risk in CRC family members and considered in clinical practice.KEYWORDS: hereditary CRC, APC gene, MSH gene, Bayesian analysis