ACTB Research Articles

MYCT-1 Gene Expression in Patients with Gastric Cancer: an Ex Vivo Study.

Ploidy, p53, bcl-2, and c-myc genes are associated with gastric cancer. Myc target 1 (MYCT1) gene is an oncogenic gene and is associated with cancer progression through different signal transduction pathways identifying the corresponding genes The objective of the study was to evaluate the association between MYCT1 gene expression and gastric cancer. Real-time polymerase chain reaction (RT-PCR), western blot analysis, cell growth study, and TUNEL assay were performed for the human gastric cancer cell lines and human embryonic kidney cell line. β-Actin gene preferred as a control in RT-PCR. The ratio of MYCT1 gene expression to β-actin gene expression less than 0.5 was considered as downregulation. Using SDS-PAGE MYCT1 gene expression was measured in western blot analysis. Cells with and without the MYCT1 gene were incubated in 35 mm plates with 10% fetal bovine serum in the cell growth study. TUNEL assay was performed to detect the effect of the MYCT1 gene on the apoptosis of cells. The ratio of MYCT1 gene expression to β-actin gene expression was 0.47 ± 0.01 and 0.76 ± 0.01 for human gastric cancer cell lines and human embryonic kidney cell lines, respectively. MYCT1 gene expression was downregulated in the human gastric cancer cell lines than human embryonic kidney cell line (p < 0.001). MYCT1 gene decreased cell growth (p = 0.041) during 6 days of incubation study of cells. TUNEL assay showed only the fluorescence of PI in BGC823 cells without the MYCT1 gene. MYCT1 gene expression was downregulated in the human gastric cancer cell lines, and MYCT1 gene accelerates the apoptotic process.

Relative expression levels of growth hormone gene and growth rate in Indian major carp species.

The phenomenon of growth is a leading factor for aquaculture success. The uneven growth of major Indian carps (Labeo rohita, Catla catla, and Cirrhinus mrigala) is a serious issue in fish culture from an economic point of view. The growth hormone (GH) gene is crucial for selection in commercially cultivated fish species for better growth and production. Indian major carp (L. rohita, C. catla, and C. mrigala) are commonly cultured in Pakistan. The GH expression was examined using qPCR to understand growth in fish species better. Muscle tissue samples (n=480) from 160 individuals of the same age were collected from three species (L. rohita, C. catla, and C. mrigala). Individuals were divided into two groups (high-weight and low-weight groups), cultured under normal conditions. The housekeeping gene β-actin validated GH expression in fast and slow-growing fishes from the same species. Results showed that GH expression varies across species and fish specimens that overweight their counterpart feature have higher GH expression. A selection for overweight fish in the aquaculture breeding systems is preferable as those fish could inherit their genomics to the future cohort, enhancing production, and commercial profit for farmers. Comprehensive research about different growth genes and the environmental aspects that influence fish growth is mandatory. No work has been reported regarding the growth gene analysis of fish from Pakistan. This report was Pakistan's first and baseline study regarding growth analysis of main culturable fish species at the molecular level.

Abstract 18748: Prevention of Skeletal Muscle Atrophy and Fibrosis in Congestive Heart Failure by Skeletal Myoblast-Targeted Overexpression of Angiotensin II Type 2 Receptor

Introduction: We have previously shown that angiotensin II (Ang II) type 2 receptor (AT2R) positively regulates skeletal myoblast differentiation. Systemic angiotensin II (Ang II) increase or congestive heart failure suppress AT2R expression, likely contributing to the development of skeletal muscle atrophy and fibrosis associated with these conditions. Hypothesis: We hypothesized that AT2R overexpression in skeletal myoblasts could prevent skeletal muscle atrophy and fibrosis associated with congestive heart failure (CHF). Approach: Two mouse models, systemic Ang II infusion and left anterior descending coronary artery (LAD) ligation were used in this study. Myoblast differentiation, muscle atrophy and muscle fibrosis were assessed by analyzing gene expressions (e.g. myogenin and atrogin-1), myofiber cross sectional area, and the collagen deposition (picrosirius red staining). Primary myoblasts were isolated from mouse hindlimb muscles. Myoblast-specific AT2R-overexpressing transgenic mice were generated by crossing Pax7 CreER/+ mice (tamoxifen-inducible cre recombinase is expressed under Pax7 promoter) and CAG-stop flox -AT2R mice (AT2R is expressed under CMV early enhancer element, chicken β-actin gene promoter upon cre-mediated excision of floxed stop codon). Results: AT2R expression was robustly increased during myoblast differentiation both in vitro and in vivo . siRNA-mediated AT2R knockdown significantly suppressed myoblast differentiation in vitro and skeletal muscle regeneration in vivo . In both Ang II infusion and LAD ligation models, AT2R induction during myoblast differentiation was significantly suppressed, associated with reduced muscle regeneration and increased atrophy and fibrosis. Myoblast-specific AT2R overexpression in vivo restored muscle regenerative capacity, and prevented muscle atrophy and fibrosis. Conclusions: AT2R induction in skeletal myoblast is suppressed in CHF, possibly via increased Ang II. Decreased AT2R expression and its downstream signaling in myoblast lead to lowered muscle regenerative capacity and increased muscle atrophy and fibrosis. Restoration of myoblast AT2R expression could lead to a novel therapy to prevent muscle atrophy and fibrosis in CHF condition.

Title: MLLT1 Is Required to Maintain Normal B-Lineage Hematopoiesis and Mitochondrial Function

MLLT1 (aka ENL) is an epigenetic reader protein that binds to crotonylated/acetylated lysine residues of histone 3 through its N-terminal YEATS domain. Multiple different positive and negative regulatory complexes compete for binding to the MLLT1 C-terminal domain. MLLT1 has been implicated in leukemic stem cell survival in MLL-rearranged cell lines but its role in normal hematopoiesis has not been investigated. Mllt1 is expressed in multiple bone marrow cell subsets, with high expression levels in multipotent and B-lineage progenitors. We generated conditional Mllt1 knockout ( Mllt1 del) mice and observed a B-lineage defect in bone marrow of Mllt1 deleted mice compared to control Mllt1 fl/fl mice. Mitochondrial activity has been linked to hematopoietic lineage fate decision choices. We performed seahorse mitochondrial stress test on lineage negative (stem and progenitor) bone marrow cells and found that lineage negative cells of Mllt1 del mice have lower mitochondrial activity (as a measure of oxygen consumption rate) than control Mllt1 fl/fl mice. In addition, we observed mitochondrial mass (as a quantitative measure of ratio of mtDNA gene CytB / nDNA gene ActB) to be decreased in lineage negative cells of Mllt1 del mice when compared to control Mllt1 fl/fl mice. Surface marker labeling was used to sort more defined subpopulations from bone marrow of Mllt1 del and Mllt1 fl/fl mice. We observed decreased mitochondrial mass in MPP4s and MEPs of Mllt1 del mice when compared to control Mllt1 fl/fl mice. Flow cytometry-based cell surface labeling to distinguish different bone marrow progenitors, plus mitochondria staining using MitoTracker Green (for mitochondrial mass) and MitoTracker Red CMXRos (for mitochondrial membrane potential) showed altered mitochondrial function of B-lineage progenitors in Mllt1 del mice when compared to control Mllt1 fl/fl mice. Thus, Mllt1 is essential for normal mitochondrial function, which may be necessary to maintain specific bone marrow hematopoietic subpopulations. Ongoing studies include gene expression profiling of sorted lymphoid progenitor populations and analysis of direct Mllt1 target genes that contribute to the altered phenotype in Mllt1 del mice.

An Advanced Multiplex Real-Time Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Rapid and Reliable Detection of Porcine Epidemic Diarrhea Virus and Porcine Internal Positive Control.

For rapid and reliable detection of porcine epidemic diarrhea virus (PEDV) from pig clinical samples, a multiplex, real-time, reverse transcription loop-mediated isothermal amplification (mqRT-LAMP) was developed using two sets of primers and assimilating probes specific to the PEDV N gene and the Sus scrofa β-actin gene, which was used as an endogenous internal positive control (EIPC) to avoid false-negative results. The assay specifically amplified both target genes of PEDV and EIPC in a single reaction without any interference but did not amplify other porcine viral nucleic acids. The limit of detection was 10 copies/μL, 100-fold lower than that of a reverse transcription-polymerase chain reaction (RT-PCR) and equivalent to that of quantitative/real-time RT-PCR (qRT-PCR). This assay has high repeatability and reproducibility with coefficients of variation < 4.0%. The positive signal of the mqRT-LAMP assay was generated within 25 min, demonstrating advantages in rapid detection of PEDV over RT-PCR or qRT-PCR assay, which require at least 2 h turnaround times. In clinical evaluation, the detection rate of PEDV by mqRT-LAMP assay (77.3%) was higher than that of RT-PCR assay (69.7%), and comparable to qRT-PCR (76.8%) with almost 100% concordance (kappa value 0.98). The developed mqRT-LAMP assay can serve as an advanced alternative method for PEDV diagnosis because it has high sensitivity and specificity, rapidity, and reliability even in resource-limited laboratories.

Assessment of virus and Leptospira carriage in bats in France.

With over 1,400 species worldwide, bats represent the second largest order of mammals after rodents, and are known to host major zoonotic pathogens. Here, we estimate the presence of pathogens in autochthonous bat populations. First, we set out to check our samples for PCR amplification efficiency by assessing the occurrence of inhibited PCR reactions from different types of bat samples with amplifying the housekeeping gene β-actin. Second, we investigated the presence of five targeted pathogens in a French bat population using PCR. We targeted viral RNA of Canine distemper virus, Alphacoronavirus, Lyssavirus, Rotavirus and bacterial Leptospira DNA. To do so, we screened for these viruses in bat faecal samples as well as in oropharyngeal swab samples. The presence of Leptospira was assessed in urine, kidney, lung and faecal samples. Results showed a frequency of inhibited reactions ranging from 5 to 60% of samples, varying according to the sample itself and also suspected to vary according to sampling method and the storage buffer solution used, demonstrating the importance of the sampling and storage on the probability of obtaining negative PCR results. For pathogen assessment, rotavirus and alphacoronavirus RNA were detected in Myotis myotis, Myotis daubentonii, Myotis emarginatus and Rhinolophus ferrumequinum bats. Rotaviruses were also detected in Barbastella barbastellus. The presence of alphacoronavirus also varied seasonally, with higher frequencies in late summer and October, suggesting that juveniles potentially play an important role in the dynamics of these viruses. Leptospira DNA was detected in M. myotis and M. daubentonii colonies. The 16S rRNA sequences obtained from Leptospira positive samples showed 100% genetic identity with L. borgpetersenii. Neither canine distemper virus nor lyssavirus RNA were detected in any of the tested samples. This study is the first to show the presence of Leptospira in autochthonous French bats in addition to coronavirus and rotavirus RNA previously reported in European autochthonous bats.

Lipid modulation during IVM increases the metabolism and improves the cryosurvival of cat oocytes

This study investigated the time course of lipid accumulation during IVM and assessed the role of lipid modulators added during IVM on lipid content, nuclear maturation, oxidative stress, mitochondrial activity, gene expression, and cryosurvival of cat oocytes. First, the lipid content of immature COCs was compared to those subjected to different IVM duration times (24, 28, and 32 h). Then, the lipid content was investigated after the use of different lipid modulators [conjugated linoleic acid (CLA), forskolin (FSK), l-carnitine (LC)]. Subsequently, both the CONTROL group and MIX 18 (CLA+FSK+LC) were compared regarding nuclear maturation, mitochondrial activity, reactive oxygen 19 species (ROS), and glutathione (GSH) levels, to the expression of SDHA, GDF9, BMP15, ZAR-1, 20 PRDX1, SIRT1, and SIRT3 genes (normalized by ACTB and YWHAZ genes); and to vitrification and 21 post-warming viability assessment. When not using any lipid modulator, an increase (P < 0.05) in lipid content could be observed after 28 h of IVM. The MIX group showed the greatest (P < 0.05) reduction in oocyte lipid content after 28 h of IVM. No difference (P > 0.05) was observed in the MII rate in the CONTROL (45%) and MIX (41%) groups and in mitochondrial activity ((1.00 ± 0.35 A U vs 1.19 ± 0.14 A U). Although ROS and GSH levels were higher (P < 0.05) in MIX than in CONTROL, the redox balance (ROS/GSH) was greater (P < 0.05) in the latter (C:1.00 ± 0.20b vs M:0.26 ± 0.06 a A.U). The GDF9, HSP70, PRDX1, and SIRT1 transcripts were downregulated (P < 0.05) in MIX-oocytes, compared to the CONTROL. After vitrification, MIX (74%) presented a higher (P < 0.05) viability compared to control (53%). In conclusion, MIX can reduce the total lipid content and improve viability after cryopreservation, however, it seems to affect the oocyte metabolism in a way that still needs to be better understood in the cat biological model.

Effect of Probiotics and Dietary Levels of Protein on Growth, Muscle Quality and Expression of Some Immune Genes in the Head Kidney of Nile Tilapia (Oreochromis Niloticus)

This study evaluated the effect of dietary probiotics (P) to enhance the feed quality, growth parameters, muscle fatty acid (FA) profile, immunity, and the stimulation of immune genes in head-kidney of Nile tilapia. Healthy mixed-sex Nile tilapia (47.0 ± 2.07 g mean weight), were fed three tilapia commercial feeds; S, A, and a local feed K having 33.3, 32.4, and 13.4% protein, respectively. Dietary supplementation of Bacillus probiotic (P) into the commercial feeds resulting in six experimental feeds (S, SP, A, AP, K, and KP) were used. The non-probiotic feeds served as controls and each treatment had three replicates. Results showed that fish fed the SP feed had the highest (P&lt;0.05) final mean weight (582.7 g), daily growth rate (2.52 g/fish/day), gross fish yield (44.0 kg/m3), and best feed conversion ratio (1.9), followed by S (570.4 g, 2.46 g/fish/day, 41.1 kg/m3, and 2.09, respectively). However, these values were the lowest (P&lt;0.05) in tilapia fed KP and K (362.8 and 366.7 g, 1.48 and 1.49 g/fish/ day, 27.4 and 26.8, kg/m3, 2.16 and 2.18, respectively). The feed A and AP had intermediate values. Fish fed the SP feed had the highest (P&lt;0.05) muscle ∑n-3 fatty acid (10.5) and ∑n-3/n-6 ratio (0.75). In general, gut bacterial counts, lysozyme activity, phagocytic activity, and hemagglutination titer were the highest (P&lt;0.05) in tilapia fed the SP, followed by tilapia fed AP, but it was the lowest for tilapia fed K and the KP feeds. Probiotics significantly up regulated β-actin, IgM, IL-β1, Mx, and TNF-α immune gene in the head kidney with better response in fish fed the SP feed. It can be concluded that improvement in tilapia growth, bacterial colonization, muscle quality, immune parameters, and the up regulation of immune genes in the head kidney due to probiotic supplementation would depend on the protein level of the feed used.

The role and expression of pro/antiangiogenic factors and microRNAs in gestational hypertension and pre-eclampsia

ObjectivePre-eclampsia and gestational hypertension are two common hypertensive disorders of pregnancy with pre-eclampsia accounting for high foetal and maternal morbidity and mortality rate. These disorders have an unknown aetiology and their hypertensive and end-organ pathophysiology may present too late in pregnancy. This makes the identification of early detection and differentiation markers vital. MicroRNAs have strongly been associated with pregnancy and their imbalance has been associated with the angiogenic dysregulation seen in pre-eclampsia. This study assesses the expression of pro- and antiangiogenic factors and their corresponding microRNAs in the maternal circulation of patients with pre-eclampsia and gestational hypertension. Study designWe analyzed angiogenic factors expression (sEng, TGF-β, VEGF) normalized against housekeeping gene β-actin and microRNAs (miRs: 210, 29B, 126) normalized against miR U6, potentially associated with pre-eclampsia and gestational hypertension using the targeted qPCR technique. These analytes were examined from early-onset (<34 weeks) (EOPE) (n = 12), late-onset (>34 weeks) (LOPE) (n = 12) pre-eclampsia, gestational hypertension (GH) (n = 12) and two gestationally matched normotensive groups (NG1 and 2) (n = 12) each in South African women of African ancestry. Group comparisons of experimental vs. control groups were assessed using t-test analysis for significance and represented as fold change expression. ResultsThe relative expression in group comparisons showed significant (p < 0.05) fold change of VEGF, TGF-β, sEng and miR126 in the EOPE vs. NG1. The GH vs. NG1 exhibited significant changes in VEGF, TGF-β, miR126, miR210 and miR29B. The LOPE vs. NG2 showed significant relative expression in all the angiogenic factors (VEGF, TGF-β and sEng). The GH vs. NG2 showed significant expression in VEGF and miR29B. The LOPE vs. EOPE showed significant fold changes in VEGF and miR210. Finally, only the GH vs. EOPE showed significant differences in miR210 and miR29B (p < 0.05). ConclusionThis study provides better insights into angiogenic factors and microRNAs specificity to the subtypes of gestational hypertensive disorders in pregnancy. Relative expression analysis of angiogenic factors and microRNAs showed possible novel characteristics of gestational hypertension, and potential common molecular and pathological profiles with pre-eclampsia. Furthermore, we postulate that sEng and miR29B could be early detection markers for pre-eclampsia and gestational hypertension, respectively.

Determining the methylation status of the promoter regions of MARCH11, HOXA9, PTGDR, and UNCX genes in patients with non-small cell lung cancer

The aim of this study was to determine the methylation status of the promoter regions of MARCH11, HOXA9, PTGDR, and UNCX genes in the tumor and non-tumor lung tissue in patients with non-small cell lung cancer (NSCLC). A relative level of methylation of the promoter regions of MARCH11, HOXA9, PTGDR, and UNCX genes was determined by the quantitative methylation-specific PCR in 73 patients with NSCLC. The quantitative methylation-specific reaction was performed both for tumor tissue samples and non-tumor tissue samples of the same patient. For each of the samples, a reaction was set both by the investigated genes (MARCH11, UNCX, HOXA9, and PTGDR) and by the reference beta-actin gene (β-actin). Positive levels of methylation of the HOXA9 gene were established for 83.5 % patients; the MARCH11 gene – for 80.8 % patients; the PTGDR gene – for 68.4 % patients; the UNCX gene – for 84.9 % patients. In the study group of patients with NSCLC, significant differences were found in the relative levels of methylation of the promoter regions of MARCH11, HOXA9, PTGDR, and UNCX genes in the tumor and non-tumor lung tissue. The data suggest that hypermethylation of MARCH11, HOXA9, PTGDR, and UNCX genes may play a role in NSCLC tumor progression.

P25 Becker naevus is associated with neurocognitive abnormalities in a substantial proportion of children

Abstract Becker naevus and Becker naevus syndrome are mosaic disorders caused in 60% of cases by postzygotic variants in the gene ACTB which encodes β-actin. We observed several patients with Becker naevus and coexistent neurocognitive abnormalities and began to study this potential association. All 51 patients seen in the last 5 years with a differential diagnosis of Becker naevus in electronic and/or photographic records were identified retrospectively. Clinical notes, photographs, radiology and histopathology were reviewed. Twenty-one patients were removed due to a different or doubtful final cutaneous diagnosis. Neurocognitive diagnoses were included only when made by a clinician, all external to the Paediatric Dermatology department. All but three patients had had their neurodevelopment assessed. Nine of the 27 patients assessed (33.3%) had a neurocognitive diagnosis, and all nine were male. Two children had autistic spectrum disorder (ASD), four had attention deficit hyperactivity disorder (ADHD) and two children had both diagnoses. MRI scans had been performed in six of the nine patients, and two had abnormal findings. A very substantial proportion of this cohort of patients with a definite diagnosis of Becker naevus have an independent diagnosis of ASD or ADHD, which in line with all-cause ASD/ADHD appears to be commoner in males. This is a new description. Germline genetic disorders caused by gain- or loss-of-function variants in ACTB are associated with neurocognitive abnormalities. Genotyping is ongoing to examine any association between the neurocognitive phenotype and mosaic ACTB variants in this clinical cohort.

Hprt Serves as an Ideal Reference Gene for qRT-PCR Normalization in Rat DRG Neurons.

To identify suitable reference genes for gene expression studies in rat dorsal root ganglia (DRG) neurons. The raw cycle threshold (Ct) values of 12 selected reference genes were obtained via quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) in neurons at different developmental stages or under different treatments. Two strategies were employed to screen the most stable reference genes: the genes were ranked according to the coefficient of biological variation and further validated using geNorm and NormFinder programs. The stable and unstable reference genes were subsequently used as internal controls to assess their effects on target gene expression. All reference genes showed varying degrees of fluctuation in Ct values during the growth process of neurons or after different treatments. 18S ribosomal RNA (Rn18s) and β-actin (Actb) exhibited the most significant changes, while ubiquitin C (Ubc), hypoxanthine phosphoribosyl transferase (Hprt), and mitochondrial ribosomal protein L10 (Mrpl10) showed relatively minor changes. The most stable and unstable genes obtained by different evaluation methods varied slightly. Overall, Actb was found to be the most unstable reference gene, while Hprt was the relatively most stable reference gene. The use of unstable reference genes Actb and ankyrin repeat domain 27 (Ankrd27) as internal controls led to high variability within the control group, ultimately affecting the determination of target gene expression. In contrast, the stable reference gene Hprt had small inter-assay variation and high stability. Our observations indicate that Hprt is a proper endogenous reference gene for qRT-PCR analysis in rat DRG neurons and thus provides a critical molecular basis for the genetic characterization in neurological disorders.

Long-term follow-up and novel variant in Suleiman-El-Hattab syndrome: Expanding the genotypic and clinical spectrum of a rare neurodevelopmental disorder

Suleiman-El-Hattab syndrome (SULEHS, OMIM #618950) is an autosomal recessive multisystem developmental disorder characterized by distinctive facial appearance, global developmental delay/intellectual disability, poor expressive speech and happy demeanor. SULEHS is an ultra-rare disorder associated with biallelic loss-of-function variants of the TASP1 gene, and up-to-date, seven patients from five families have been reported in the literature. Loss of TASP1 function has been reported to alter H3K4 histone modifications and expression of TFIIA and HOX transcription factors in the SULEHS phenotype. In this report, a new patient molecularly diagnosed with SULEHS by a novel homozygous c.404-2A > G variant in the TASP1 gene is presented with the long-term follow-up. Although the majority of the patient's clinical characteristics were similar to those of previously reported SULEHS patients, this study was the first to describe some additional anomalies, such as cystic hygroma, increased nuchal thickness, coarctation of the aorta, pulmonary stenosis, pulmonary sequestration anomaly, chronic constipation, encephalomalacia, and aggressive behavior. Because of the remarkable similarities between the clinical features of Baraitser-Winter syndrome (BRWS) and the patient, BRWS was considered the most likely diagnosis before the molecular diagnosis. Network analysis also supported that the interaction of the SULEHS-associated TASP1 gene with the BRWS-associated ACTB and ACTG1 genes through common intermediate molecules. Overall, despite the existence of differences in clinical features, inheritance patterns, and underlying pathophysiology between BRWS and SULEHS, both diseases could be considered in the differential diagnosis due to the high clinical similarities, including the dysmorphic features, growth parameters, neurodevelopmental phenotype, neurological problems, and multisystem involvement. Additionally, this report could contribute to a better understanding of the genotypic and clinical features of SULEHS by describing a novel pathogenic variant and new clinical features, such as prenatal manifestations.

Expression profiling of laccase and β-glucan synthase genes in Pleurotus ostreatus during different developmental stages.

Pleurotus ostreatus, commonly known as the oyster mushroom, is a saprophytic fungus with many applications in biotechnology and medicine. This mushroom is a rich source of proteins, polysaccharides, and bioactive compounds that have been shown to possess anticancer, antioxidant, and immunomodulatory properties. In this study, we investigated the expression profile of laccase (POXA3) and β-glucan synthase (FKS) genes during different developmental stages in two strains of P. ostreatus. Cultural and morphological studies of the two strains were studied. DMR P115 strain recorded faster mycelial growth compared to the HUC strain. However, both strains produced white, thick fluffy mycelial growth with radiating margin. Morphological characteristics of the mushroom fruiting body were also higher in the DMR P115 strain. The expression of these genes was analyzed using quantitative real-time PCR (qPCR) and the results were compared to those of the reference gene β-actin. The expression of laccase (POXA3) was higher in the mycelial stage of DMR P115 and HUC strains indicating its role in the fruiting body development and substrate degradation. The expression of β-glucan synthase (FKS) was upregulated in the mycelium and mature fruiting body of the DMR P115 strain. In contrast, there was only significant upregulation in the mycelial stage of the HUC strain, which indicates its role in cell wall formation and the immunostimulatory properties of that strain. The results deepen the understanding of the molecular mechanism of the fruiting body development in P. ostreatus and can be used as a foundation for future lines of research related to strain improvement of P. ostreatus.

Comparative Analysis of the Expression Profiles of Pathogenesis-Related Genes in Tomato Systemically Infected with Tobacco Mosaic and Cucumber Mosaic Viruses

In this study, we used RT-qPCR to examine how PR genes were expressed in model tomato (Solanum lycopersicum L.) plants that had been infected with TMV or CMV. Under greenhouse conditions, the indirect ELISA data showed that both viruses were detected for the first time at 6 dpi. Then, the levels of accumulation increased very quickly, reaching a peak of 15 dpi. During the course of the study (1–15 dpi), the Delta CT, NormFinder, BestKeeper, and GeNorm software tools revealed that the β-actin gene was the most informative reference gene in the virally infected tomato tissues. For both the TMV- and CMV-infected tomato plants, the transcriptional expression levels of most tested genes changed between activation and repression, especially in the first 12 dpi. Compared to mock-inoculated plants, the expression levels of PR-1 were induced at all time intervals except at 8 dpi for CMV and at 6, 7, and 8 dpi for TMV infection. Conversely, the greater activation and accumulation of both viruses were associated with the greater up-regulation of PR-2 at 8 dpi, with relative expression levels of 7.28- and 5.84-fold for TMV and CMV, respectively. The up-regulated expression of PR-3, PR-4, and PR-7 was shown at 4 dpi. In contrast, the PR-5 gene was inhibited in TMV at 1 dpi until 9 dpi, and the induction of this gene at 10 dpi increased by 1.72-fold, but PR-5 was observed to up-regulate the expression of CMV at 1 dpi. This study provides the first valuable information on the comparative transcriptional levels of these tomato genes between TMV and CMV infections.

Identification of leaf chloroplast-specific promoter to efficiently control of Colorado potato beetle with reduced dsRNA accumulation in potato tubers.

By expressing double-stranded RNA (dsRNA) in potato plastids targeting the β-Actin (ACT) gene of the Colorado potato beetle (CPB), transplastomic plants can trigger the beetle's RNA interference response to kill the CPB larvae. High expression of dsACT driven by rrn16 promoter (Prrn) in the leaf chloroplasts of transplastomic plants confers strong resistance to CPB. However, there are still residual amounts of dsRNA in the tubers, which are unnecessary for CPB control and may raise a potential food exposure issue. In order to reduce dsRNA accumulation in the tubers while maintaining stable resistance to CPB, we selected two promoters (PrbcL and PpsbD) from potato plastid-encoded rbcL and psbD genes and compared their activities with Prrn promoter for dsRNA synthesis in the leaf chloroplasts and tuber amyloplasts. We found that the dsACT accumulation levels in leaves of transplastomic plants St-PrbcL-ACT and St-PpsbD-ACT were significantly reduced when compared to St-Prrn-ACT, but they still maintained high resistance to CPB. By contrast, a few amounts of dsACT were still accumulated in the tubers of St-PrbcL-ACT, whereas no dsACT accumulation in tubers was detectable in St-PpsbD-ACT. We identified PpsbD as a useful promoter to reduce dsRNA accumulation in potato tubers while maintaining the high resistance of the potato leaves to CPB. © 2023 Society of Chemical Industry.

Investigation of gene expression levels in thyroid tissues of rats treated with Wi-Fi electromagnetic wave (2.4–3 GHz Wi-Fi RF-EMF)

In this study, it was aimed to examine the effects of wireless waves applied to rats in the thyroid tissue by evaluating gene expression levels on 12 determined gene regions. 20 healthy 16-week-old Wistar albino female rats weighting 200–220 g were used. In the experiments, two groups, control and experimental groups, were formed, and ten female rats were used in each group. While WI-FI electromagnetic field was applied to the experimental group of these rats, nothing was applied to the control group. Many different gene regions, including beta catenin, beta-actin, GAPDH, L3B, HIF 1-Alpha, GSk-3B, TCF, WNT7a, WNT10a, WNT2, Beclin 1 and Beclin 2, ATG5 and ATG12, were investigated. ACTB and GAPDH gene primers were used as a house keeping gene. The fold change values were calculated statistically from the data obtained from the gene expression results. Compared to the control group, it was observed that the experimental group had significant increases in gene expression levels of many gene regions investigated in the study (P<0.05). Among the study groups, 12 different genes such as beta catenin, beta-actin, GAPDH, L3B, HIF 1-Alpha, GSk-3B, TCF, WNT7a, WNT10a, WNT2, Beclin 1 and Beclin 2, ATG5 and ATG12, which we have determined according to the literature, are known to be related to autophagy and oxidative stress. When the expression levels were investigated, it was determined that the expression coefficients of all the genes studied in the wnt/ß catenin pathway in the experimental group of rats exposed to the Wi-Fi EM field, except for ATG5 and ATG12, were quite high, and there was significant differences between the groups.

What to Do if the qPCR Test for SARS-CoV-2 or Other Pathogen Lacks Endogenous Internal Control? A Simple Test on Housekeeping Genes.

Some of the products for the molecular diagnosis of infections do not have an endogenous internal control, and this is necessary to ensure that the result is not a false negative. The aim of the project was to design a simple low-cost RT-qPCR test that can confirm the expression of basic metabolism proteins, thus confirming the quality of genetic material for molecular diagnostic tests. Two successful equivalent qPCR assays for the detection of the GADPH and ACTB genes were obtained. The course of standard curves is logarithmic, with a very high correlation coefficient R2 within the range of 0.9955-0.9956. The reaction yield was between 85.5 and 109.7%, and the detection limit (LOD) with 95% positive probability was estimated at 0.0057 ng/µL for GAPDH and 0.0036 ng/µL for ACTB. These tests are universal because they function on various types of samples (swabs, cytology, etc.) and can complement the diagnosis of SARS-CoV-2 and other pathogens, as well as potentially oncological diagnostics.

Development of a Cell Line Containing the Chimeric ETV6-NTRK3 Gene. The Search for Mutations of the Tyrosine Kinase Chimeric Domain That Cause Resistance to Larotrectinib.

The development, registration, and further use of entrectinib and larotrectinib for the treatment of tumors resulting from oncogenic stimulation of chimeric neurotrophin receptors (TRK) attracted much interest to the mechanisms of tumor cells resistance to TRK inhibitors during treatment. In the presented study, a cell line carrying the chimeric gene ETV6-NTRK3 (HFF-EN) was created on the basis of human fibroblasts. The transcription level of the chimeric ETV6-NTRK3 gene in HFF-EN was comparable to the transcription level of the household ACTB gene, the expression of the ETV6-NTRKA protein was confirmed by immunoblotting. A comparison of the dose-effect curves of fibroblasts and HFF-EN cells showed a ~38-fold increase in the sensitivity of HFF-EN to larotrectinib. To obtain a cell model of the resistance to larotrectinib in NTRK-dependent cancer, we used cell passages with a gradually increasing concentration of larotrectinib and obtained six resistant clones. p.G623E c.1868G>A mutation was found in five clones, and p.R582W c.1744C>T mutation, previously not described as a resistance mutation, was found in one clone showing significantly less resistance. These results can be further used for more complete understanding of the mechanisms of the resistance to TRK inhibitors and for the development of new drugs.

Abstract 2201: Effect of opioid and cannabinoid receptors gene expression on survival of patients with colorectal cancer

Abstract Introduction: Colorectal cancer (CRC) is one of the leading causes of cancer death in the world. CRC patients are often treated with opioid (e.g., morphine, or synthetic opioid piritramide) analgesia for pain, nausea, and side effects of chemotherapy. These analgesics act through opioid and cannabinoid receptors, which pathways are involved in tumor progression and metastases and thus can negatively affect the survival of patients. In our previous studies, we analyzed the presence of circulating tumor cells (CTCs) in two groups of CRC patients. One group received morphine analgesia (MA) and second piritramide analgesia (PA). We found that MA associated with increased CTCs levels and significantly shorter cancer-specific survival (CSS). These findings lead to further analysis of the opioid and cannabinoid receptors tumor gene expressions. We expected that the different expression of specific receptors in tumor tissue makes a difference between the effects of MA and PA on patients´ survival. Methods: Gene expression of opioid receptors µ (OPRM), κ (OPRK), δ (OPRD), nociceptin (OPRL) and cannabinoid receptors 1 and 2 (CB1, CB2) was analyzed in RNA purified from tumor tissues in 131 patients with CRC. Expression of markers was detected using real-time RT-PCR on LightCycler 1536 from Roche. Β-actin gene expression was used for gene expression normalization. Specific cut-off values were calculated for each marker using maxstat R software, ver. 3.3.1. Relationship between expression of receptors in tumor tissue and patients’ survival was analyzed using COX regression, Kruskal-Wallis/ANOVA test, and Kaplan-Meier method. Results: In total, 118 patients (45 females and 73 males, average age 68 years), of clinical stage-III were analyzed. It was found that MA in CRC patients was associated with significantly shorter cancer-specific survival (CSS; p=0,027). The univariate survival analysis revealed that CRC patients with high OPRM tumor tissue gene expression had significantly longer overall survival (OS; p=0.017) and patients with CB2 gene expression had significantly longer disease-free survival (DFS; p=0,029). All other tested opioid receptors expressions had no statistically significant effect on patients’ survival. Conclusion: The diverse opioid receptors’ expressions on tumor and immune cells can affect the survival of cancer patients treated with opioid analgesia. Morphine perioperative analgesia seems to shorten the CCS in CRC patients in our study. However, further research is required to elucidate the effect of opioids on different cell populations. Acknowledgement: This study was supported by Ministry of Health of the Czech Republic [NV18-03-00470]; Palacky University Olomouc [LF 2022_012]; European Regional Development Fund [ENOCHCZ.02.1.01/0.0/0.0/16_019/0000868) and European Union - Next Generation EU (EXCELES LX22NPO5102). Citation Format: Monika Vidlarova, Josef Srovnal, Emil Berta, Sona Gurska, Alona Rehulkova, Pavla Kourilova, Marian Hajduch. Effect of opioid and cannabinoid receptors gene expression on survival of patients with colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2201.