Relative Expression Of Target Genes Research Articles

MiR-301b-3p promotes breast cancer development through inhibiting the expression of transforming growth factor-beta receptor 2.

Breast cancer (BC) is a serious health threat to the patients. The present work explored the mechanism of miR-301b-3p and transforming growth factor-beta receptor 2 (TGFBR2 ) in affecting BC progression. The miR-301b-3p-inhibitor and si-TGFBR2 solution were added to the DEME/F12 medium to culture the BC and normal breast epithelial cell lines to prepare negative control, miR-301b-3p-IN and miR-301b-3p-IN+si-TGFBR2 in the two types of cell lines. The relative expression of target genes and the interference effect were analyzed by quantitative real-time PCR (qRT- PCR). Cell viability was detected applying cell counting kit-8 (CCK-8) assay. Transwell and wound healing assay were conducted to evaluate the invasion and migration of BC cells after miR-301b-3p inhibition. Additionally, cell apoptosis and the expression STAT protein were measured by flow cytometry and Western blot, respectively. The qRT-PCR results showed that miR-301b-3p were high-expressed but the level of TGFBR2 was significantly inhibited in BC cells. The miR-301b-3p-inhibitor significantly downregulated the expression of miR-301b-3p and upregulated that of TGFBR2. Meanwhile, inhibition of miR-301b-3p suppressed the cell viability, invasion, and migration of BC cells, which, however, were restored by the inhibition of TGFBR2. MiR-301b-3p conferred anti-apoptosis ability to BC cells, while TGFBR2 promoted apoptosis of BC cells through producing an antagonistic effect with miR-301b-3p. We found that miR-301b-3p played a crucial role in the phosphorylation of STAT1 and STAT3 to promote BC progression. The present findings demonstrated that miR-301b-3p played a crucial role in promoting BC cell growth, invasion and migration and anti-apoptosis, and that targeting TGFBR2 could inhibit the tumor-promoting effect of miR-301b-3p.

Herbicide paraquat dichloride augments the expression of the c-abl-noxa-apoptotic pathway, instigating ovarian atresia in edible fish, Channa punctatus.

Ovarian cells of animals undergo atresia to regulate egg numbers and their release. However, unrestrained atretic activity decreases the chances of oocyte maturity and further process, ultimately resulting in a reduced population. Edible fishes are a critical source of nutrition and it is required to regulate their population. The application of herbicides at fish production sites is a threat to their population by altering female reproductive activity. In concern to this, here, this 60-day study investigates herbicide paraquat dichloride (PD)-induced atresia in ovarian cells of Channa punctatus (C. punctatus). Further, 96-h LC50 of PD for C. punctatus was calculated as 58.40mg/L. At the end of each exposure period, an increase in intracellular ROS in blood cells and activities of SOD and CAT in ovary tissue were observed in PD-treated fish with increasing concentration. The frequency of micronuclei in blood cells subsequently increased with the highest PD concentration. A significant (p < 0.05) increase in the relative expression of target genes was observed at the 30th and 60th day of the sampling periods in the fish treated with the highest concentration of PD. The histological study confirmed the occurrence of atretic ovary cells in PD-exposed fish. From the results it can be concluded that PD enhances the uncontrolled death of ovarian cells and has consequences of impairing the ovary health and reproductive potential of fish, hence reducing the fish population.

Modulation of Human Bone Marrow Mesenchymal Stem Cells (BMMSC) by Nigella Sativa and Trigona Honey: An in vitro Study

Maintenance of bone marrow mesenchymal stem cells (BMMSC) numbers is crucial for tissue repair and regeneration since adult stem cells are often limited in number, expansion capacity and lifespan. It is believed that certain types of foods are beneficial in the preservation and stimulation of stem cells throughout life. Black seeds and stingless bee honey are among the widely investigated functional food with general health promoting benefit. We aim to explore the proliferative, anti-apoptosis and anti-senescence effect of Nigella sativa and Trigona honey on BMMSC. Cell proliferation was evaluated using MTT assay. We performed flow cytometric analysis to verify stem cell surface markers while quantitative PCR was done to measure the relative expression of target genes. Results showed favourable concentration dependent enhancement of cell proliferation by 1 to 10 ug/mL Nigella sativa and 0.097% to 0.195% Trigona honey. Observation on BMMSC’s morphology and surface markers expression revealed no alteration on BMMSC stemness properties. In addition, gene expression analysis supported that there was a significant (P&lt;0.05) increase in proliferation related gene, β-Catenin, and lower expression of apoptotic- and senescence-related gene, BAX and p21, respectively. These preliminary findings suggest a promising effect of Nigella sativaand Trigona honey on stem cell proliferation.

Isolation, cloning, and tissue distribution and functional analysis of ShP-glycoprotein in the freshwater crab Sinopotamon henanense exposed to Cd and Cd-QDs

P-glycoprotein (Pgp), a member of ATP binding cassette (ABC) transporter family, can extrude toxic substances out of cells by mediating multi-xenobiotic resistance (MXR) in aquatic organisms, however, its regulation and association with MXR are still unclear. In this work, the genetic information of Pgp in freshwater crab Sinopotamon henanense (ShPgp) was revealed for the first time. ShPgp with a total of 4488 bp was cloned and analyzed, which includes 4044 bp open reading frame, 353 bp 3′ untranslated region, and 91 bp 5′ untranslated region. The recombinant ShPGP were expressed in Saccharomyces cerevisiae and taken for SDS-PAGE and western blot analysis. ShPGP was widely expressed in the midgut, hepatopancreas, testis, ovary, gill, hemocytes, accessory gonad and myocardium of the crabs studied. The images of immunohistochemistry indicated that ShPgp was mainly distributed in the cytoplasm and cell membrane. When the crabs were exposed to cadmium or cadmium containing quantum dots (Cd-QDs), not only the relative expression of ShPgp mRNA and the protein produced were enhanced, but also the MXR activity and ATP contents. The relative expression of target genes related to energy metabolism, detoxification and apoptosis was also determined in the carbs exposed to Cd or Cd-QDs. The results showed that bcl-2 was significantly down-regulated, while other genes were up-regulated except PPAR (not affected). However, when the Shpgp in treated crabs was interfering by knockdown technique, their apoptosis and the expression of proteolytic enzyme genes and transcription factors MTF1 and HSF1 were also elevated, while the expression of apoptosis inhibiting and fat metabolism genes were compromised. Based on the observation, we concluded that MTF1 and HSF1 were involved in gene transcription regulation of mt and MXR, respectively, while PPAR had limited regulatory effect on those genes in S. henanense. NF-κB may play a negligible role in the process of apoptosis in testes induced by cadmium or Cd-QDs. However, the detail information regarding Pgp involvement in SOD or MT, and its association with apoptosis during xenobiotics insults remain to be explored.

Rewired Metabolism Caused by the Oncogenic Deregulation of MYC as an Attractive Therapeutic Target in Cancers.

MYC is one of the most deregulated oncogenes on multiple levels in cancer. As a node transcription factor, MYC plays a diverse regulatory role in many cellular processes, including cell cycle and metabolism, both in physiological and pathological conditions. The relentless growth and proliferation of tumor cells lead to an insatiable demand for energy and nutrients, which requires the rewiring of cellular metabolism. As MYC can orchestrate all aspects of cellular metabolism, its altered regulation plays a central role in these processes, such as the Warburg effect, and is a well-established hallmark of cancer development. However, our current knowledge of MYC suggests that its spatial- and concentration-dependent contribution to tumorigenesis depends more on changes in the global or relative expression of target genes. As the direct targeting of MYC is proven to be challenging due to its relatively high toxicity, understanding its underlying regulatory mechanisms is essential for the development of tumor-selective targeted therapies. The aim of this review is to comprehensively summarize the diverse forms of MYC oncogenic deregulation, including DNA-, transcriptional- and post-translational level alterations, and their consequences for cellular metabolism. Furthermore, we also review the currently available and potentially attractive therapeutic options that exploit the vulnerability arising from the metabolic rearrangement of MYC-driven tumors.

DSN enzyme recognition initiated rolling circle amplification (DiRCA) for accurate miRNA detection in immune response and immune repair after trauma

Recent studies have reported that miRNA plays an important role in immune response and immune repair after trauma. By regulating the expression of related target genes, miRNA regulates the production, proliferation, development and immune response of immune cells. Therefore, it is in urgent demand to develop an novel method for miRNA analysis. Rolling circle amplification (RCA), as an attractive isothermal signal amplification strategy, has been widely utilized in constructing miRNA detection assays. However, accurate and sensitive miRNA quantitative determination remains a huge challenge for RCA based approaches. Herein, we propose a DSN enzyme based signal cycle initiated Rolling Circle Amplification assay (DiRCA) for sensitive and accurate miRNA detection. In DiRCA, target miRNA unfolds hairpin structure probe in the detection scaffold, forming a RNA–DNA duplex. DSN enzyme is utilized to specifically digest the DNA sequence in RNA–DNA duplex, releasing miRNA to form a signal cycle; its capability to distinguish one base pair mismatch in RNA–DNA duplex endows DiRCA a high selectivity. Meanwhile, DSN enzyme based cleavage initiates RCA, transcribing G-rich sequences for signal generation. Based on the DSN assisted signal cycle and RCA, DiRCA shows a low limit of detection of 0.43 fM and a superior capability in selectively detecting mismatched miRNA sequences, showing a promising prospect in the early-diagnosis of disease.

Effects of Dietary Blend of Algae Extract Supplementation on Growth, Biochemical, Haemato-Immunological Response, and Immune Gene Expression in Labeo rohita with Aeromonas hydrophila Post-Challenges

In this study, the effects of a mixed algal blend (Chlorella vulgaris, Euglena viridis, and Spirulina platensis) at different levels were evaluated on growth, hematological immune responses, and expression of immune genes in Labeo rohita against post-challenges of Aeromonas hydrophila. Fish samples were fed a diet containing different levels of mixed blend algal (0, 0.01, 0.02, 0.04 and 0.08% of basal diet). At the end of the feeding period, the fish were challenged with A. hydrophila and fish mortality was recorded over a 14-days period. To evaluate the serum biochemical (albumin, globulin), hematological parameters (Hb, RBC and WBC) and immune parameters (neutrophil activity, lysozyme activity, myeloperoxidase activity, antiprotease activity, ceruloplasmin activity, and bactericidal activity), as well as the expression of immune genes (NKEF-B, Lysozyme C and G, TNF α, TLR22, β2M, and β-actin), fish were sampled on Day 7, 14, 21 and 28. Fish were challenged with virulent A. hydrophila 30 days post-feeding and mortalities were recorded over 30 days post-infection. Results demonstrate that fish fed with a mixed algal blend showed that total body weight gain, specific growth rate, total serum protein, globulin, total hemoglobin content, white blood cells, neutrophil, lysozyme, bactericidal, myeloperoxidase, and antiprotease activity in dietary algae blended application was higher than in the control (p &lt; 0.05). According to the results, relative expression of target genes showed significant increases of 0.02 to 0.04% in the treatment group compared to the control group (p &lt; 0.05). At the end of the 30-day exposure to A. hydrophila, the fish that received the mixed algal blend had a significantly higher rate of survival than the control group, with the highest survival rate recorded in the 0.02% mixed algal blend (p &lt; 0.05). According to the effective results of the mixed algal blend on stimulating the immune system and increasing fish resistance to A. hydrophila, it is recommended to use 0.02 to 0.04% of this mixed algal blend in rohu, L. rohita diets.

Evaluation of Reference Genes for Real-Time Quantitative PCR Analysis in Tissues from Bumble Bees (Bombus Terrestris) of Different Lines.

Bumble bees are important alternative pollinators and model insects due to their highly developed sociality and colony management. In order to better understand their molecular mechanisms, studies focusing on the genetic and molecular aspects of their development and behavior are needed. Although quantitative real-time polymerase chain reaction (qRT-PCR) can be used to quantify the relative expression of target genes, internal reference genes (which are stably expressed across different lines and tissues) must first be identified to ensure the accurate normalization of target genes. In order to contribute to molecular studies on bumble bees, we used Bombus terrestris to determine the expression stability of eight reference genes (β-actin (ACT), Arginine Kinase (AK), Phospholipase A2 (PLA2), Elongation factor 1 alpha (EF-1), Ribosomal proteins (S5, S18, S28) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)) in five different lines and several tissues (ovary, thorax, fat body, and head) using RT-qPCR procedures and four analysis programs (RefFinder, NormFinder, BestKeeper, and geNorm). In general, the S28, S5, and S18 ribosomal protein genes and the PLA2 and EF-1 genes showed the highest stability and were therefore identified as suitable reference genes for the bumble bee species and their defined lines and tissues. Our results also emphasized the need to evaluate the stability of candidate reference genes for any differently designed lines and tissue conditions in bumble bee species.

Selection and Identification of a Reference Gene for Normalizing Real-Time PCR in Mangos under Various Stimuli in Different Tissues

Real-time quantitative polymerase chain reaction (Real-Time PCR) is a rapid, highly sensitive, and highly specific technique, which is widely used to determine the relative expression of target genes in plants. It plays an indispensable role in searching for stable reference genes in different species. However, no suitable reference genes for real-time PCR normalization have been reported in mangos. In this study, 10 candidate reference genes were obtained from the ‘Carabao’ genome, and their expression stability under seven abiotic stresses (MeJA, Mannitol, NaCl, SA, ABA, heat, and cold) and in four different tissues (root, stem, leaf, and fruit) was rated using four professional reference gene scoring software packages (geNorm, NormFinder, BestKeeper, and RefFinder). The results indicated that the stability of the 10 selected genes varied significantly under different experimental conditions; moreover, TUBB is more stable than the other candidate reference genes and can be used as a suitable reference gene, since it was always ranked as one of the top three in different tissues and under multiple conditions, according to the comprehensive ranking. To ensure the applicability of the identified reference genes, the relative expression levels of Chalcone synthase 1 (CHS-1) and Chalcone synthase 2 (CHS-2) were used to confirm the accuracy of the results. The evaluation of the stability of multiple reference genes will facilitate the future accurate quantification of target genes by real-time PCR in mangos.

Pengaruh Askorbat dan Hipoksia Terhadap Penghambatan Migrasi Sel Serta Penurunan Ekspresi Gen HIF-1α dan Twist pada Lini Sel Kanker Payudara MCF-7

Hypoxia at cellular level can induce the particular changes of certain gene expression. HIF-1α stability can support cancer cell migration through the regulation of TWIST expression. Hypoxic condition will maintain HIF-1α accumulation due to a decreased hydroxylases activity. The expression of HIF-1α is known to be reduced transcriptionally and translationally by increasing ascorbate under hypoxic. However, the role of ascorbate in inhibiting cell migration and its effect on the downregulation of HIF-1α and TWIST gene expression have not been clearly understood. Therefore, this study is aimed to observe the effect of ascorbate and hypoxia that regulate the inhibition of HIF-1α and TWIST. Scratch assay method was used to observe cell migration. RT-PCR method was used to detect HIF-1α and TWIST gene expression. The results showed that ascorbate and hypoxia inhibit MCF-7 cell migration within 34 hours. Under normoxic, ascorbate with the concentration of 0.40 mM (IC50) suppressed cell migration significantly with an average migration rate (Vm) relative to control of 1,28 µm/hour and a percentage of wound area closure of 19,37%. Under hypoxic, ascorbate with the concentration of 1,20 mM (IC75) inhibited cell migration with an average Vm of 0,93 µm/hour and reduced the percentage of wound area up to 14,2%. Relative expression of target genes under hypoxic was compared with normoxic condition. The results showed that ascorbate with the concentration of 1,20 mM (IC75) downregulated HIF-1α gene expression up to ±6,97x and TWIST gene up to ±3,25x under hypoxic condition. In summary, ascorbate and hypoxia can inhibit cell migration and downregulate HIF-1α and TWIST gene expression in MCF-7 cell.

Screening and stability analysis of reference genes in fasting caecotrophy model in rabbits.

The selection and validation of stably expressed reference genes is key for accurately quantifying the mRNA abundance of genes under different treatments. In the rabbit model of fasting caecotrophy, reports about the selection of stable reference genes are not available. This study aims to screen suitable reference genes in different tissues (including uterus, cecum, and liver) of rabbits between control and fasting caecotrophy groups. RT-qPCR was used to analyze the expression levels of eight commonly used reference genes (including GAPDH, 18S rRNA, B2M, CYP, HPRT1, β-actin, H2afz, Ywhaz), and RefFinder (including geNorm, NormFinder, and BestKeeper) was used to analyze the expression stability of these reference genes. Our results showed that the most stable reference genes were different in different tissues and treatments. In the control and fasting caecotrophy groups, CYP, GAPDH and HPRT1 were proven to be the top stable reference genes in the uterus, cecum, and liver tissues, respectively. GAPDH and Ywhaz were proven to be the top two stable reference genes among uterus, cecum, and liver in both control and fasting caecotrophy groups. Our results indicated that the combined analysis of three or more reference genes (GAPDH, HPRT1, and Ywhaz) are recommended to be used for RT-qPCR normalization in the rabbit model of fasting caecotrophy, and that GAPDH is a better choice than the other reference genes for normalizing the relative expression of target genes in different tissues of fasting caecotrophy rabbits.

Phase 1 study of CB-103, a novel first-in-class inhibitor of the CSL-NICD gene transcription factor complex in human cancers.

3020 Background: CB-103 selectively inhibits the CSL-NICD interaction leading to down-regulation of CSL-NICD mediated oncogenic pathway activation downstream of NOTCH receptor/ligand signaling, and has shown potent anti-cancer activity as single agent and in combination with targeted/chemotherapies in preclinical models. The aim of this dose escalation/expansion phase 1/2a study is to assess safety, maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D), preliminary activity, pharmacokinetics and pharmacodynamics of CB-103. Methods: Eligible were adult patients (pts) with advanced or recurrent selected solid tumors. Tumor tissue, where available, was retrospectively tested for NOTCH pathway activating mutations and surrogate tissues were evaluated for gene expression of related target genes. CB-103 was given orally in 28 days cycles at escalating doses until disease progression or toxicity. In a dose confirmatory cohort, NOTCH activation will be prospectively assessed to determine eligibility. Results: Forty-one pts (19 adenoid cystic carcinoma (ACC), 16 colorectal and 4 breast cancer, 2 prostate cancer) were assigned to increasing dose levels starting from 15mg once daily (OD). Median age was 55 years (range 25-76). Median number of prior lines of therapy was 2 (range 0-7). Thirty-two pts in 8 escalation groups completed the 28-day DLT window. One DLT (asymptomatic grade (G) 3 GGT increase) was observed at the highest dose (600mg). Related treatment emergent adverse events (AE) occurring in &gt;10% of pts were nausea (24%), diarrhea (20%), dyspepsia (15%), fatigue (12%) and vision blurred (12%), all G 1/2. No discontinuations occurred due to treatment-related AEs. The MTD has not been reached. Several pts reported vision changes that improved over time and were fully reversible after stopping the drug. Median time on treatment for all pts was 52 days (range 5-249). Best response was stable disease (SD). For ACC pts, preliminary median PFS was 21.7 weeks (95% confidence interval (CI) 13.7-22.4 weeks) and disease control rate (DCR) was 79% at week 8 and 58 % at week 20. Three pts with ACC harboring activating NOTCH alterations had radiologically confirmed stable disease (SD) &gt; 6 months. Importantly, in 3 pts with NOTCH positive disease a temporary stop of tumor growth was observed. One pt showed a reduction in size of a liver lesion up to 25% before progression due to new lesions. Mechanistically, strong on-treatment downregulation of NOTCH target genes was observed. The dose of 600mg CB-103 OD was declared the RP2D. Conclusions: CB-103 is the first drug to effectively control the CSL-NICD transcription complex. CB-103 is well tolerated in pts with advanced tumors and, as expected on the basis of mechanistic studies, in the absence of the typical toxicities associated with Notch targeting GSIs or mABs. The RP2D has been established for advancing clinical development into phase 2. Clinical trial information: NCT03422679.

Investigation of MRP1 and ABCG2 Gene Expression in Chronic Myeloid Leukemia (CML) Patients

Background: This study evaluated and compared the quantitative expression of multidrug resistance-associated protein 1 (MRP1) and ATP-binding cassette subfamily G member 2 (ABCG2), two Multidrug Resistance (MDR) related genes, in 30 CML patients and 27 normal subjects. Methods: Total RNA was isolated from peripheral blood mononuclear cells (MNCs) using the Trizol reagent. Then cDNAs were synthesized. Gene expression was quantified using Real-Time PCR System. The relative expression of target genes was calculated using the 2-ΔΔCt method. Results: High expression of MRP1 and ABCG2 mRNAs were detected in the patient group. Intra-group comparisons also revealed increased expression of ABCG2 in Accelerated Phase (AP)-Blastic Crisis (BC) patients compared to Chronic Phase (CP) patients. At the same time, the increased expression of MRP1 in AP-BC patients was not statistically significant. Conclusion: Considering the broad spectrum of ATP Binding Cassette (ABC) transporter superfamily substrates, they can play an essential role in cell fate determination. High expression of MRP1 and ABCG2 genes can result in the efflux of therapeutic agents and subsequent reduction in their intracellular concentration. This mechanism finally protects cells from the therapeutic effects of medications. On the other hand, these transporters can export growth factors out of the cell. Such exported molecules may have a growth-inducing effect on adjacent cells. These are the possible mechanisms for the participation of MRP1 and ABCG2 genes in conferring drug resistance to CML cells.

Phenotypic characterization of frontal cortex microglia in a rat model of post-traumatic stress disorder.

IntroductionPost‐traumatic stress disorder (PTSD) is an anxiety disorder induced by psychologically traumatic events. Using a rat model, this study aimed to determine whether psychological trauma alters relative expression between pro‐inflammatory and anti‐inflammatory markers in microglia. To meet this goal, expression of genes encoding i‐NOS, arginase, TNF‐α, interleukin‐10, CD74, and Mannose Receptor C was analyzed on multiple days following trauma exposure.MethodsSingle‐prolonged stress (SPS) was used to model PTSD in male Sprague‐Dawley rats. Twenty‐four rats (12 Controls and 12 SPS‐exposed) were sacrificed on Days 1, 3, and 7 post‐SPS. Twenty‐four (12 Controls and 12 SPS‐exposed) additional rats were exposed to classical fear conditioning on Day 7, and fear extinction on Days 8, 9, 10, 15, 16, and 17. Freezing behavior was measured to assess fear resolution. Microglial isolates were collected from the frontal cortex, and RNA was extracted. Changes in relative expression of target genes were quantified via RT‐PCR.ResultsSPS rats showed significant decreases in IL‐10 and TNF‐α expression and increases in the i‐NOS:Arginase and TNF‐α:IL‐10 ratios compared to Controls on Day 1, but not on Day 3 or Day 7 for any of the dependent variables. Day 17 SPS rats showed a significant decrease in IL‐10 expression and an increase in the TNF‐α:IL‐10 ratio, further characterized by a significant inverse relationship between IL‐10 expression and fear persistence.ConclusionPsychological trauma impacts the immunological phenotype of microglia of the frontal cortex. Consequently, future studies should further evaluate the mechanistic role of microglia in PTSD pathology.

Identification of reliable reference genes for expression studies in maternal reproductive tissues and foetal tissues of pregnant cows.

The relationship between the conceptus and the maternal uterine environment is crucial for the successful establishment and maintenance of pregnancy in cattle. Gene expression analysis of the conceptus and maternal reproductive tissues is a favourable method to assess the embryonic maternal interaction. The reliability of the commonly used method reverse transcription-quantitative polymerase chain reaction (RT-qPCR) depends on proper normalization to stable reference genes (RGs). The objective of this study was to determine the expression stability of 10 potential RGs in maternal reproductive tissues and foetal tissues, and to analyse the effect of RG selection on the calculation of the relative expression of target genes. The expression stability of 10 potential RGs was analysed in eight different tissues from three pregnant dairy cows. Three programs-GeNorm, NormFinder and Bestkeeper-were used to identify the best RGs. According to all three programs, the most stable RG was CNOT11, whereas the least stable RGs were GAPDH and HPRT1. GeNorm analysis showed that a combination of five RGs (SDHA, PPIA, CNOT11, RPS9 and RPL19) was necessary for appropriate data normalization. However, NormFinder analysis indicated that the combination of CNOT11 and PPIA was the most suitable. When target genes were normalized to these RGs, the relative expression of the Radical S-adenosyl methionine domain containing 2 gene was not affected by the choice of RGs, whereas a large difference was observed in the expression profile of the Nuclear erythroid2-related factor 2 gene between the most stable and least stable RGs. The results indicate that careful selection of RGs is crucial under different conditions, especially for target genes with relatively small fold changes. Furthermore, the results provide useful information for the selection of RGs for evaluating genes affecting bovine reproduction.

Reference gene selection for qRT-PCR analysis of season- and tissue-specific gene expression profiles in the honey bee Apis mellifera

Honey bees are both important pollinators and model insects due to their highly developed sociality and colony management. To better understand the molecular mechanisms underlying honey bee colony management, it is important to investigate the expression of genes putatively involved in colony physiology. Although quantitative real-time PCR (qRT-PCR) can be used to quantify the relative expression of target genes, internal reference genes (which are stably expressed across different conditions) must first be identified to ensure accurate normalisation of target genes. To identify reliable reference genes in honey bee (Apis mellifera) colonies, therefore, we evaluated seven candidate genes (ACT, EIF, EF1, RPN2, RPS5, RPS18 and GAPDH) in samples collected from three honey bee tissue types (head, thorax and abdomen) across all four seasons using three analysis programmes (NormFinder, BestKeeper and geNorm). Subsequently, we validated various normalisation methods using each of the seven reference genes and a combination of multiple genes by calculating the expression of catalase (CAT). Although the genes ranked as the most stable gene were slightly different on conditions and analysis methods, our results suggest that RPS5, RPS18 and GAPDH represent optimal honey bee reference genes for target gene normalisation in qRT-PCR analysis of various honey bee tissue samples collected across seasons.

SAT-587 Molecular Markers of Beige Adipose Following ESR1 Knockdown in the Mediobasal Hypothalamus of Adult Female Rhesus Monkeys

Our studies in female marmoset monkeys show that the ablation of ovarian estradiol (E2) production fails to alter energy homeostasis or body fat accumulation. Peripheral E2 may therefore not play a crucial role in metabolic regulation in female primates. shRNA-mediated knockdown of ESR1 expression in the hypothalamic ventromedial nucleus (VMN) in adult female rodents, however, induces obesity and suggests ESR1 is a hypothalamic target for E2 regulation of energy homeostasis, and likely mediates thermogenesis in brown/beige adipose depots. In female primates, including humans, the hypothalamic estrogen receptor mediating metabolic regulation is unknown. To test the hypothesis that ESR1 mediates female primate regulation of energy homeostasis, 11 ovary intact, adult female rhesus macaques, pair housed with female peers, received five 12µl MRI-guided MBH infusions into the rostral-to-caudal extent of both right and left VMN. Each infusion comprised a gadolinium contrast agent and ~3–4 x 1010 adeno-associated virus 8 (AAV8) particles containing either an shRNA specific for ESR1 (n=6, ERaKD) or scrambled shRNA (n=5, control). Mid-surgery MRI scans identified targeting accuracy. ~ 1.5 yrs following AAV8 infusion, pronounced gain in BMI enabled conversion of 83% of ERaKD females to overweight/obese compared to 20% of controls (p=0.08). Percent increase in BMI remained intermittently greater (p<0.05) than controls thereafter. Adipose depots were harvested at necropsy ~2.5–3 yrs following treatment. Total RNA was isolated using the Qiagen AllPrep DNA/RNA/miRNA Universal kit. RNA was reverse transcribed with High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). All quantitative real-time PCR (qRT-PCR) were performed on a StepOnePlus System using Power SYBR Green master mix (Applied Biosystems). Primer sequences were designed using NCBI Primer-Blast. Expression of TATA-box binding protein (TBP) was used as the internal control housekeeping gene. The relative expression of target genes was measured using the comparative cycle threshold (Ct) method with results expressed as target mRNA expression relative to TBP using the formula 2^-delta Ct. Upper body beige adipose represents an organ system in primates, including humans, involved in thermogenesis. Axillary beige adipose depots in ERaKD females, however, did not exhibit significantly diminished gene expression for selected markers of beige adipocytes, including PAT2, CD137 and C/EBPβ, compared to control females. More crucially, thermogenically relevant UCP1 expression also did not differ between ERaKD females and controls. Taken together, these results suggest that knockdown of VMN ESR1 in adult female monkeys, while inducing modest weight gain after 1.5 years, may not markedly alter beige adipose gene expression of initially selected thermogenically relevant genes.

Effects of feeding with red algae (Laurencia caspica) hydroalcoholic extract on antioxidant defense, immune responses, and immune gene expression of kidney in rainbow trout (Oncorhynchus mykiss) infected with Aeromonas hydrophila

In this study, the effects of hydroalcoholic extract of red algae (Laurencia caspica) at different levels were evaluated on antioxidant defense, innate immune responses, and expression of immune genes in rainbow trout (Oncorhynchus mykiss). A total of 750 rainbow trout (39.43 ± 0.44 g) were randomly assigned to five treatments with three replicates (50 fish per replicate) in 15 ponds and reared for 8 weeks. Fish samples were fed a diet containing different levels of algal extract (0, 5, 10, 15, and 20 g extract per kg of basal diet). At the end of feeding period, the fish were challenged with Aeromonas hydrophila and fish mortality was recorded over a 10-day period. To evaluate the antioxidant and immune parameters, fish were sampled at day 0, end of weeks 4 and 8, and after bacterial challenge. Expression of immune genes (IL-1β, lysozyme II, Complement C3, and TNFα) was also examined after bacterial challenge. Results indicated that application of algal powder in fish diet improved lysozyme activity, complement system, and immunoglobulin levels of extract fed-fish compared to control group (p < .05). The activities of antioxidant enzymes (catalase, glutathione peroxidase, and superoxide dismutase) were affected by the algal extract, with increasing levels in algal extract treatments compared to control fish (p < .05). According to the results, relative expression of target genes showed significant increases in 1.5% treatment compared to the other and control groups (p < .05). At the end of 10-day fish exposure to A. hydrophila, fish survived significantly higher in treatments received the algal extract than the control group, with the highest survival rate recorded in 1.5% algal extract (p < .05). According to the effective results of L. caspica extract on stimulating the immune system and increasing fish resistance to A. hydrophila, it is recommended to use 1.5% of this algal extract in rainbow trout diets.

Allograft inflammatory factor-1 supports macrophage survival and efferocytosis and limits necrosis in atherosclerotic plaques

Background and aimsAllograft inflammatory factor-1 (AIF1) has been characterized as a pro-inflammatory molecule expressed primarily in the monocyte/macrophage (MP) lineage and positively associated with various forms of vascular disease, including atherosclerosis. Studies of AIF1 in atherosclerosis have relied on mouse models in which AIF1 was overexpressed in either myeloid or smooth muscle cells, resulting in increased atherosclerotic plaque burden. How physiologic expression of AIF1 contributes to MP biology in atherogenesis is not known. MethodsEffects of global AIF1 deficiency on atherosclerosis were assessed by crossing Aif1−/− and ApoE−/− mice, and provoking hyperlipidemia with high fat diet feeding. Atherosclerotic plaques were studied en face and in cross section. Bone marrow-derived MPs (BMDMs) were isolated from Aif1−/− mice for study in culture. ResultsAtherosclerotic plaques in Aif1−/−;ApoE−/− mice showed larger necrotic cores compared to those in ApoE−/− animals, without change in overall lesion burden. In vitro, lack of AIF1 reduced BMDM survival, phagocytosis, and efferocytosis. Mechanistically, AIF1 supported activation of the NF-κB pathway and expression of related target genes involved in stress response, inflammation, and apoptosis. Consistent with this in vitro BMDM phenotype, AIF1 deficiency reduced NF-κB pathway activity in vivo and increased apoptotic cell number in atherosclerotic lesions from Aif1−/−;ApoE−/− mice. ConclusionsThese findings characterize AIF1 as a positive regulator of the NF-κB pathway that supports MP functions such as survival and efferocytosis. In inflammatory settings such as atherosclerosis, these AIF1-dependent activities serve to clear cellular and other debris and limit necrotic core expansion, and may oppose lesion destabilization.

Dysregulation of microRNA146a-5p expression in systemic lupus erythematosus females: Diagnostic potential and association with ocular manifestations

BackgroundMicro-ribonucleic acids (miRNA) are key regulators for the expression of related target genes and their aberrant expression has a substantial role across many pathways within autoimmunity. Aim of the workTo evaluate the expression of miRNA-146a-5p in systemic lupus erythematosus (SLE) patients and to assess its relation to the disease activity and various disease parameters with specific focus on the ocular manifestations. Patients and methods42 female SLE patients and 39 matched healthy individuals were enrolled in this study. The serum level of miRNA146a-5p was evaluated by a quantitative real-time polymerase chain reaction (PCR) (Taqman technology). ResultsThe mean age of the patients was 29 ± 9.1 years. The serum level of miRNA146a-5p was significantly higher in SLE cases (3.21 ± 4.47 fold change) compared to the controls (1.54 ± 1.66 fold change) (p = 0.001). A significant negative association was found between the serum level of miRNA146a-5p and C4 level (p = 0.018). Expression levels of miRNA146a-5p were significantly decreased in patients with photosensitivity (p = 0.006). Regarding ocular manifestations, a lower level of miRNA146a-5p was detected in patients suffering from dry eyes, cataract, keratitis and drusen compared to patients without and a higher level was found in those suffering from retinopathy. Serum miRNA146a-5p at a cut off value of 1.36 could discriminate patients from controls (95%CI: 0.61–0.84; p = 0.001) at a sensitivity of 69% and specificity of 64.1%. ConclusionThese findings suggest that miR-146a-5p could be a potential diagnostic biomarker in SLE and may implicate a functional role in the pathophysiology of the ocular manifestations in the disease.